Carbon Fixation Systems in Plants and Algae

ABSTRACT

Provided are heterologous nucleic acid constructs, vectors and methods for elevating cyclic electron transfer activity, improving carbon concentration, and enhancing carbon fixation in C3 and C4 plants, and algae, and producing biomass or other products from C3 or C4 plants, and algae, selected from among, for example, starches, oils, fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, and organic acids, as well as transgenic plants produced thereby. These methods and transgenic plants and algae encompass the expression, or overexpression, of various combinations of genes that improve carbon concentrating systems in plants and algae, such as bicarbonate transport proteins, carbonic anhydrase, light driven proton pump, cyclic electron flow regulators, etc.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/358,331, titled “Improved Carbon Fixation Systems in Plants andAlgae”, filed on Mar. 3, 2019, which claims priority to U.S. patentapplication Ser. No. 15/411,854, entitled “Improved Carbon FixationSystems in Plants and Algae”, filed on Jan. 20, 2017, and issued on Mar.3, 2019 as U.S. Pat. No. 10,233,458, which is a continuation ofInternational Patent Application No. PCT/US2015/041617, entitled“Improved Carbon Fixation Systems in Plants and Algae”, filed on Jul.22, 2015, which claims priority to and the benefit of the filing of U.S.Provisional Patent Application No. 62/027,354, entitled “Carbon FixationSystems in Plants and Algae”, filed on Jul. 22, 2014, and thespecification and claims thereof are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants Nos.DOE-CECO Prime No: DE-AR0000202, Sub No: 21018-N; DOE-CABS Prime No:DE-SC0001295, Sub No: 21017-NM NSF EF-1219603, NSF No:1219603. The U.S.government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 5, 2017, isnamed 040517_NMC0001-101-US_Sequence_Listing_ST25.txt and is 286 KB insize.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

COPYRIGHTED MATERIAL

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BACKGROUND

A major factor limiting photosynthetic efficiency is the competitiveinhibition of CO₂ fixation by oxygen, due to lack of specificity of theenzyme RuBisCO. Incorporation of oxygen by RuBisCO is thefirst-dedicated step in photorespiration, a pathway that respires CO₂,compounding photosynthetic inefficiency. Overall, photorespirationreduces photosynthetic productivity by as much as 50% [1]. To date,attempts to engineer reduced oxygenase activity in RuBisCO have beenlargely unsuccessful.

Significantly, the cyanobacteria, eukaryotic microalgae, and C4 plantshave evolved mechanisms to reduce photorespiration by concentrating CO₂near RuBisCO, competitively inhibiting oxygenase activity and leading tosubstantial increases in yield and water use efficiency per unit carbonfixed. However, carbon concentrating systems (CCMs) are not operationalin the vast majority of plant species (i.e., C3 plants).

Attempts to reconstitute functional CCMs in C3 plants have beenpreviously attempted by us and others, mainly focusing on engineeringpathways that are directly involved in facilitating CO₂ transport intoleaf chloroplasts. Note, for example, PCT International Publication WO2012/125737; Sage and Sage (2009) Plant and Cell Physiol. 50(4):756-772;Zhu et al. (2010) J Interg. Plant Biol. 52(8):762-770; Furbank et al.(2009) Funct. Plant Biol. 36(11):845-856; Weber and von Caemmerer (2010)Curr. Opin. Plant Biol.; Price (2013) J. Exp. Bot. 64(3):753-68; andU.S. Patent Application Publication No. 2013/0007916 A1.

However, ATP and NADPH production through light harvesting and electrontransfer steps must be coordinated with carbon assimilation andadditional energy requiring steps including CCM systems to preventphotoinhibition and to improve growth. Additionally, assimilatory fluxand storage rates can limit carbon fixation due to feedback inhibitionwhen sink demand is not matched to source capacity [2].

Thus, there is a critical need to improve plant productivity throughintegrated systems engineering approaches that balance source/sinkinteractions with energy and reductant production to developenergy-requiring, artificial CCMs that can effectively mimic those foundin nature.

BRIEF SUMMARY OF THE INVENTION

Accordingly, in response to this need, the present disclosure providesmethods for elevating cyclic electron transfer activity, improvingcarbon concentration, and enhancing carbon fixation in C3 and C4 plants,and algae, and producing biomass or other products from C3 or C4 plants,and algae, selected from among, for example, starches, oils, fattyacids, lipids, cellulose or other carbohydrates, alcohols, sugars,nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, andorganic acids, as well as transgenic plants produced thereby. Thesemethods and transgenic plants and algae encompass the expression, oroverexpression, of various combinations of genes that improve carbonconcentrating systems in plants and algae, such as bicarbonate transportproteins, carbonic anhydrase, light driven proton pump, cyclic electronflow regulators, etc. Thus, among its various embodiments, the presentdisclosure provides the following:

A first embodiment of the present invention provides for a transgenicplant or alga, comprising within its genome, and expressing oroverexpressing, a combination of heterologous nucleotide sequencesencoding an ATP dependent bicarbonate anion transporter localized to theplasma membrane and a cyclic electron transfer modulator protein. Thecyclic electron transfer modulator protein may be selected from a PGRL1protein (for example SEQ ID NO:3), a PGR5 protein (for example SEQ IDNO:1), a leaf FNR1 protein (for example SEQ ID NO:96), a leaf FNR2protein (for example SEQ ID NO:97), a Fd1 protein (for example SEQ IDNO:95), or any combination thereof and for example the ATP dependentbicarbonate anion transporter localized to the plasma membrane may be aHLA3 protein (for example SEQ ID NO:77). The transgenic plant or algadescribed may further comprise within its genome, and expressing oroverexpressing the heterologous nucleotide sequence encoding abicarbonate anion transporter protein localized to the chloroplastenvelope. The transgenic plant or alga described herein may furthercomprise within its genome, and expressing or overexpressing theheterologous nucleotide sequence a carbonic anhydrase protein. In apreferred embodiment, the cyclic electron transfer modulator protein isa PGR5 protein, in another preferred embodiment the cyclic electrontransfer modulator protein is Fd1 protein, in yet another preferredembodiment, in still another preferred embodiment the cyclic electrontransfer modulator protein is leaf FNR1, in a further preferredembodiment the cyclic electron transfer modulator protein is PGRL1. In apreferred embodiment the heterologous nucleotide sequences of thetransgenic plant or alga encode i) a PGR5 protein, and a HLA3 protein;or ii) a PGR5 protein, a HLA3 protein and a PGRL1 protein ora PGR5protein, a HLA3 protein, and a LCIA protein or a PGR5 protein, a HLA3protein, a PGRL1 protein, a LCIA protein, and a BCA or HCA2 protein. Inanother preferred embodiment the heterologous nucleotide sequences thetransgenic plant or alga of wherein encode a PGR5 protein, a HLA3protein, a LCIA protein and a BCA or optionally a HCA2 protein. Thetransgenic plant or alga as described wherein the PGR5 protein has anamino acid sequence at least 80% identical to SEQ ID NO:1; the HLA3protein has an amino acid sequence at least 80% identical to SEQ IDNO:77; the PGRL1 protein has an amino acid sequence at least 80%identical to SEQ ID NO:3; the LCIA protein has an amino acid sequence atleast 80% identical to SEQ ID NO:18; and/or the BCA protein has an aminoacid sequence at least 80% identical to SEQ ID NO:21. Alternatively, thesequence identity/sequence similarity is about 75%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to those specifically disclosedwhich includes for example proteins without a transit peptide sequenceand the functional protein.

A second embodiment provides for a transgenic plant or alga, comprisingwithin its genome, and expressing or overexpressing, a combination ofheterologous nucleotide sequences encoding:

LCIA protein and BCA protein or HCA protein is provided. In a preferredembodiment the heterologous nucleotide sequences encode transgenic plantor alga wherein the LCIA protein has an amino acid sequence at least 80%identical to SEQ ID NO:18; and/or the BCA protein has an amino acidsequence at least 80% identical to SEQ ID NO:21 and the HCA protein hasan amino acid sequence at least 80% identical to SEQ ID NO:19.Alternatively, the sequence identity/sequence similarity is about 75%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to thosespecifically disclosed which include for example proteins without atransit peptide sequence and the functional protein.

A third embodiment provides for a transgenic plant or alga, comprisingwithin its genome, and expressing or overexpressing, a combination ofheterologous nucleotide sequences encoding an ATP dependent bicarbonateanion transporter localized to the plasma membrane (for example SEQ IDNO:77), a bicarbonate anion transporter localized to the chloroplastenvelope (for example SEQ ID NO:18), a carbonic anhydrase, aproteorhodopsin protein targeted to thylakoid membranes (for example SEQID NO:98), and a R carotene monooxygenase protein (for example SEQ IDNO:100). In another preferred embodiment the proteorhodopsin comprises achloroplast transit peptide selected from among a psbX stop-transfertrans-membrane domain fused to its C-terminus, a DNAJ transit peptide, aCAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide.In another preferred embodiment the β-carotene monooxygenase isexpressed under the control of a promoter selected from among an ethanolinducible gene promoter and a green tissue/leaf-specific promoterselected from among CAB and rbcS. The proteorhodopsin may comprise anamino acid substitution selected from among L219E/T206S, M79T, and M79Y,and combinations thereof.

The carbonic anhydrase of the first, second, or third embodiment may bea BCA or optionally a HCA2 protein. The bicarbonate anion transporterlocalized to the chloroplast envelope of the first, second and thirdembodiment may be a LCIA protein. The ATP dependent bicarbonate aniontransporter localized to the plasma membrane of the first and thirdembodiments may be HLA3.

A fourth embodiment provides for a method of making a transgenic plantor alga of a first embodiment wherein said method comprises expressing,or overexpressing, in a C3 plant, a C4 plant, or an alga, a combinationof heterologous nucleotide sequences encoding an ATP dependentbicarbonate anion transporter localized to the plasma membrane and acyclic electron transfer modulator protein. The cyclic electron transfermodulator protein may be selected from a PGRL1 protein, a PGR5 protein,a FNR1 protein, a FNR2 protein (leaf-form isotopes), a Fd1 protein, orany combination thereof and wherein the ATP dependent bicarbonate aniontransporter localized to the plasma membrane is a HLA3 protein. Theheterologous nucleotide sequences of the fourth embodiment furtherencoding a bicarbonate anion transporter protein localized to thechloroplast envelope for example the bicarbonate anion transporterprotein is LCIA. Additionally, the heterologous nucleotide sequencesencode a carbonic anhydrase protein for example a BCA protein oroptionally a HCA2 protein. In a preferred embodiment the cyclic electrontransfer modulator protein is a PGR5 protein and optionally a PGRL1protein and or combination thereof.

A fifth embodiment provides a method of making a transgenic plant oralga as described in a second embodiment, wherein said method comprisesexpressing, or overexpressing, in a C3 plant, a C4 plant, or an alga, acombination of heterologous nucleotide sequences encoding a LCIA proteinand a BCA protein or optionally a HCA protein.

A sixth embodiment provides a method of making a transgenic plant oralga of a third embodiment wherein said method comprises expressing, oroverexpressing, in a C3 plant, a C4 plant, or an alga, a combination ofheterologous nucleotide sequences encoding an ATP dependent bicarbonateanion transporter localized to the plasma membrane, a bicarbonate aniontransporter, a carbonic anhydrase, a proteorhodopsin protein targeted tothylakoid membranes, and a R carotene monooxygenase protein. In apreferred embodiment the proteorhodopsin comprises a chloroplast transitpeptide selected from among a psbX stop-transfer trans-membrane domainfused to its C-terminus, a DNAJ transit peptide, a CAB transit peptide,a PGR5 transit peptide, and a psaD transit peptide. In another preferredembodiment the β-carotene monooxygenase is expressed under the controlof a promoter selected from among an ethanol inducible gene promoter anda green tissue/leaf-specific promoter selected from among CAB and rbcS.In a preferred embodiment the proteorhodopsin comprises an amino acidsubstitution selected from among L219E/T206S, M79T, and M79Y, andcombinations thereof. In another preferred embodiment the ATP dependentbicarbonate anion transporter localized to the plasma membrane is HLA3.

The transgenic plant of an embodiment disclosed herein may be a C3 plantor a C4 plant such as a transgenic oilseed plant or a transgenic foodcrop plant which may include the genera Brassica (e.g., rapeseed/canola(Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea),Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis),coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard otherthan Arabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm;palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis;Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphealanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina;Vernonia galamensis; Momordica charantia; and Crambe abyssinica, wheat,rice, maize (corn), barley, oats, sorghum, rye, and millet; peanuts,chickpeas, lentils, kidney beans, soybeans, lima beans; potatoes, sweetpotatoes, and cassavas; soybeans, corn, canola, peanuts, palm, coconuts,safflower, cottonseed, sunflower, flax, olive, and safflower; sugar caneand sugar beets; bananas, oranges, apples, pears, breadfruit,pineapples, and cherries; tomatoes, lettuce, carrots, melons,strawberry, asparagus, broccoli, peas, kale, cashews, peanuts, walnuts,pistachio nuts, almonds; forage and turf grasses; alfalfa, clover;coffee, cocoa, kola nut, poppy; vanilla, sage, thyme, anise, saffron,menthol, peppermint, spearmint and coriander and preferably wheat, riceand canola. The transgenic alga of an embodiment disclosed herein may beselected from among a Chiorella species, a Nannochioropsis species, anda Chiamydomonas species. The heterologous nucleotide sequences aredescribed in an embodiment may be codon-optimized for expression in saidtransgenic plant or alga. One aspect of the present invention providesfor a transgenic plant or alga as described in an embodiment whichexhibits enhanced CO₂ fixation compared to an otherwise identicalcontrol plant grown under the same conditions for example wherein CO₂fixation is enhanced in the range of from about 10% to about 50%compared to that of an otherwise identical control plant grown under thesame conditions.

A fourth embodiment provides for a part of said transgenic plant or algaof any embodiment described herein. For example, the part of saidtransgenic plant may be selected from among a protoplast, a cell, atissue, an organ, a cutting, an explant, a reproductive tissue, avegetative tissue, biomass, an inflorescence, a flower, a sepal, apetal, a pistil, a stigma, a style, an ovary, an ovule, an embryo, areceptacle, a seed, a fruit, a stamen, a filament, an anther, a male orfemale gametophyte, a pollen grain, a meristem, a terminal bud, anaxillary bud, a leaf, a stem, a root, a tuberous root, a rhizome, atuber, a stolon, a corm, a bulb, an offset, a cell of said plant inculture, a tissue of said plant in culture, an organ of said plant inculture, a callus, propagation materials, germplasm, cuttings,divisions, and propagations.

A fifth embodiment provides for a progeny or derivative of saidtransgenic plant or alga of any embodiment described herein. Forexample, the progeny or derivatives may be selected from among clones,hybrids, samples, seeds, and harvested material thereof and may beproduced sexually or asexually.

Another embodiment of the present invention provides a method ofelevating CET activity in a C3 plant, C4 plant, or alga wherein saidmethod comprises expressing, or overexpressing, in a C3 plant, a C4plant, or an alga, a combination of heterologous nucleotide sequencesencoding an ATP dependent bicarbonate anion transporter localized to theplasma membrane and cyclic electron transfer modulator protein.

Yet another embodiment provides a method of enhancing carbon fixation ina C3 plant, C4 plant, or alga wherein said method comprises expressing,or overexpressing, in a C3 plant, a C4 plant, or an alga, a combinationof heterologous nucleotide sequences encoding an ATP dependentbicarbonate anion transporter localized to the plasma membrane and acyclic electron transfer modulator protein.

Yet another method provides for a method of producing biomass or otherproducts from a C3 plant, C4 plant, or an alga, wherein said productsare selected from among starches, oils, fatty acids, triacylglycerols,lipids, cellulose or other carbohydrates, alcohols, sugars,nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, andorganic acids wherein said method comprises expressing, oroverexpressing, in a C3 plant, a C4 plant, or an alga, a combination ofheterologous nucleotide sequences encoding an ATP dependent bicarbonateanion transporter localized to the plasma membrane and a cyclic electrontransfer modulator protein. This method further comprises growing saidplant or alga and harvesting said biomass or recovering said productfrom said plant or alga. Another aspect of the present inventionprovides for biomass or other product produced from a plant or algaselected from among starches, oils, fatty acids, lipids, cellulose orother carbohydrates, alcohols, sugars, nutraceuticals, pharmaceuticals,fragrance and flavoring compounds, and organic acids, made by a methodof any one of the method of making a transgenic plant or algaembodiments herein.

Another embodiment provides a method of elevating cyclic electrontransfer (CET) activity in a C3 plant, C4 plant, or alga wherein saidmethod comprises expressing, or overexpressing, in a C3 plant, a C4plant, or an alga, a combination of heterologous nucleotide sequencesencoding an ATP dependent bicarbonate anion transporter localized to theplasma membrane, a bicarbonate anion transporter, a carbonic anhydrase,a proteorhodopsin protein targeted to thylakoid membranes; and a Rcarotene monooxygenase protein.

Another embodiment provides a method of enhancing carbon fixation in aC3 plant, C4 plant, or alga wherein said method comprises expressing, oroverexpressing, in a C3 plant, a C4 plant, or an alga, a combination ofheterologous nucleotide sequences encoding an ATP dependent bicarbonateanion transporter localized to the plasma membrane, a bicarbonate aniontransporter, a carbonic anhydrase, a proteorhodopsin protein targeted tothylakoid membranes; and a R carotene monooxygenase protein.

Another embodiment provides for a method of producing biomass or otherproducts from a C3 plant, C4 plant, or an alga, wherein said productsare selected from among starches, oils, fatty acids, triacylglycerols,lipids, cellulose or other carbohydrates, alcohols, sugars,nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, andorganic acids wherein said method comprises expressing, oroverexpressing, in a C3 plant, a C4 plant, or an alga, a combination ofheterologous nucleotide sequences encoding an ATP dependent bicarbonateanion transporter localized to the plasma membrane, a bicarbonate aniontransporter, a carbonic anhydrase, a proteorhodopsin protein targeted tothylakoid membranes; and a R carotene monooxygenase protein. The methodfurther comprises growing said plant or alga and harvesting said biomassor recovering said product from said plant or alga.

Another embodiment provides for use of a construct comprising one ormore nucleic acids encoding

-   -   a) a PGR5 protein, and a HLA3 protein;    -   b) a PGR5 protein, a HLA3 protein and a PGRL1 protein;    -   c) a PGR5 protein, a HLA3 protein, and a LCIA protein;    -   d) a PGR5 protein, a HLA3 protein, a LCIA protein and a BCA or        HCA2 protein;    -   e) a PGR5 protein, a HLA3 protein, a PGRL1 protein and a LCIA        protein;    -   f) a PGR5 protein, a HLA3 protein, a PGRL1 protein, a LCIA        protein, and a BCA or HCA2 protein;    -   g) a PGR5 protein, a HLA3 protein, and a BCA or HCA2 protein; or    -   h) a PGR5 protein, a HLA3 protein, a PGRL1 protein, and a BCA or        HCA2 protein        for    -   i) making a transgenic plant or alga of a first embodiment;    -   ii) elevating CET activity in a C3 plant, C4 plant, or alga;    -   iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga;        or    -   iv) producing biomass or other products from a C3 plant, C4        plant, or an alga, wherein said products are selected from among        starches, oils, fatty acids, triacylglycerols, lipids, cellulose        or other carbohydrates, alcohols, sugars, nutraceuticals,        pharmaceuticals, fragrance and flavoring compounds, and organic        acids.

Another embodiment provides for use of a construct comprising one ormore nucleic acids encoding

-   -   a) a LCIA protein and a BCA or HCA2 protein;        for    -   i) making a transgenic plant or alga of a second embodiment;    -   ii) elevating CET activity in a C3 plant, C4 plant, or alga;    -   iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga;        or    -   iv) producing biomass or other products from a C3 plant, C4        plant, or an alga, wherein said products are selected from among        starches, oils, fatty acids, triacylglycerols, lipids, cellulose        or other carbohydrates, alcohols, sugars, nutraceuticals,        pharmaceuticals, fragrance and flavoring compounds, and organic        acids.

One aspect of the present invention provides for a transgenic plant oralga, comprising within its genome, and expressing or overexpressing, acombination of heterologous nucleotide sequences encoding:

1. i) a PGRL1 protein, a PGR5 protein, and a HLA3 protein; or

-   -   ii) a PGRL1 protein, a PGR5 protein, a HLA3 protein, a LCIA        protein, and a BCA or HCA2 protein; or    -   iii) a Fd1 protein, a HLA3 protein, a LCIA protein, and a BCA or        HCA2 protein; or    -   iv) a leaf FNR1 protein, a HLA3 protein, a LCIA protein, and a        BCA or HCA2 protein; or    -   v) a proteorhodopsin protein targeted to thylakoid membranes, a        HLA3 protein, a LCIA protein, a BCA or HCA2 protein, and a        β-carotene monooxygenase.

2. The transgenic plant or alga of 1, wherein said proteorhodopsincomprises a chloroplast transit peptide selected from among a psbXstop-transfer trans-membrane domain fused to its C-terminus, a DNAJtransit peptide, a CAB transit peptide, a PGR5 transit peptide, and apsaD transit peptide.

3. The transgenic plant or alga of 1 or 2, wherein said β-carotenemonooxygenase is expressed under the control of a promoter selected fromamong an ethanol inducible gene promoter and a greentissue/leaf-specific promoter selected from among CAB and rbcS.

4. The transgenic plant or alga of any one of 1-3, wherein saidproteorhodopsin comprises an amino acid substitution selected from amongL219E/T206S, M79T, and M79Y, and combinations thereof.

5. The transgenic plant of any one of 1-4, which is a C3 plant or a C4plant.

6. The transgenic plant of any one of 1-5, which is a transgenic oilseedplant or a transgenic food crop plant.

7. The transgenic oilseed plant of 6, which is selected from amongplants of the genera Brassica (e.g., rapeseed/canola (Brassica napus;Brassica carinata; Brassica nigra; Brassica oleracea), Camelina,Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis), coconut;cotton; peanut; rice; safflower; sesame; soybean; mustard other thanArabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm; palmkernel; sugarcane; castor bean; switchgrass; Borago officinalis; Echiumplantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphea lanceolata;Ricinus communis; Coriandrum sativum; Crepis alpina; Vernoniagalamensis; Momordica charantia; and Crambe abyssinica.

8. The transgenic alga of any one of 1-5, which is selected from amongChlorella sp., Nannochloropsis sp., and Chlamydomonas sp.

9. The transgenic plant or alga of any one of 1-8, wherein saidheterologous nucleotide sequences are codon-optimized for expression insaid transgenic plant or alga.

10. The transgenic plant or alga of any one of 1-9, which exhibitsenhanced CO₂ fixation compared to an otherwise identical control plantgrown under the same conditions.

11. The transgenic plant or alga of 10, wherein CO₂ fixation is enhancedin the range of from about 10% to about 50% compared to that of anotherwise identical control plant grown under the same conditions.

12. A part of said transgenic plant or alga of any one of 1-11.

13. The part of said transgenic plant of 12, which is selected fromamong a protoplast, a cell, a tissue, an organ, a cutting, an explant, areproductive tissue, a vegetative tissue, biomass, an inflorescence, aflower, a sepal, a petal, a pistil, a stigma, a style, an ovary, anovule, an embryo, a receptacle, a seed, a fruit, a stamen, a filament,an anther, a male or female gametophyte, a pollen grain, a meristem, aterminal bud, an axillary bud, a leaf, a stem, a root, a tuberous root,a rhizome, a tuber, a stolon, a corm, a bulb, an offset, a cell of saidplant in culture, a tissue of said plant in culture, an organ of saidplant in culture, a callus, propagation materials, germplasm, cuttings,divisions, and propagations.

14. Progeny or derivatives of said transgenic plant or alga of any oneof 1-11.

15. The progeny or derivatives of 14, which is selected from amongclones, hybrids, samples, seeds, and harvested material thereof.

16. The progeny of 14 or 15, which is produced sexually.

17. The progeny of 14 or 15, which is produced asexually.

Another aspect of the present invention provides for a method selectedfrom among:

18. i) making a transgenic plant or alga of any one of 1-11;

ii) elevating CET activity in a C3 plant, C4 plant, or alga;

iii) enhancing carbon fixation in a C3 plant, C4 plant, or alga; and

iv) producing biomass or other products from a C3 plant, C4 plant, oralga, wherein said products are selected from among starches, oils,fatty acids, triacylglycerols, lipids, cellulose or other carbohydrates,alcohols, sugars, nutraceuticals, pharmaceuticals, fragrance andflavoring compounds, and organic acids,

wherein said method comprises expressing, or overexpressing, in a C3plant, a C4 plant, or an alga, a combination of heterologous nucleotidesequences encoding:

-   -   a) a PGRL1 protein, a PGR5 protein, and a HLA3 protein; or    -   b) a PGRL1 protein, a PGR5 protein, a HLA3 protein, a LCIA        protein, and a BCA or HCA2 protein; or    -   c) a Fd1 protein, a HLA3 protein, a LCIA protein, and a BCA or        HCA2 protein; or    -   d) a leaf FNR1 protein, a HLA3 protein, a LCIA protein, and a        BCA or HCA2 protein; or    -   e) a proteorhodopsin protein targeted to thylakoid membranes, a        HLA3 protein, a LCIA protein, a BCA or HCA2 protein, and a        β-carotene monooxygenase.

19. The method of 18, wherein step iv) further comprises growing saidplant or alga and harvesting said biomass or recovering said productfrom said plant or alga.

20. The method of 18 or 19, wherein said proteorhodopsin comprises achloroplast transit peptide selected from among a psbX stop-transfertrans-membrane domain fused to its C-terminus, a DNAJ transit peptide, aCAB transit peptide, a PGR5 transit peptide, and a psaD transit peptide.

21. The method of any one of 18-20, wherein said β-carotenemonooxygenase is expressed under the control of a promoter selected fromamong an ethanol inducible gene promoter and a greentissue/leaf-specific promoter selected from among CAB and rbcS.

22. The method of any one of 18-21, wherein said proteorhodopsincomprises an amino acid substitution selected from among L219E/T206S,M79T, and M79Y, and combinations thereof.

23. The method of any one of 18-22, wherein said transgenic plant is aC3 plant, a C4 plant, or an alga.

24. The method of any one of 18-23, wherein said transgenic plant is atransgenic oilseed plant or a transgenic food crop plant.

25. The method of 24, wherein said transgenic oilseed plant is selectedfrom among plants of the genera Brassica (e.g., rapeseed/canola(Brassica napus; Brassica carinata; Brassica nigra; Brassica oleracea),Camelina, Miscanthus, and Jatropha; Jojoba (Simmondsia chinensis),coconut; cotton; peanut; rice; safflower; sesame; soybean; mustard otherthan Arabidopsis; wheat; flax (linseed); sunflower; olive; corn; palm;palm kernel; sugarcane; castor bean; switchgrass; Borago officinalis;Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima; Cuphealanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina;Vernonia galamensis; Momordica charantia; and Crambe abyssinica.

26. The method of any one of 18-23, wherein said alga is selected fromamong Chlorella sp., Nannochloropsis sp., and Chlamydomonas sp.

27. The method of any one of 18-26, wherein said heterologous nucleotidesequences are codon-optimized for expression in said transgenic plant oralga.

28. The method of any one of 18-27, wherein said transgenic plant oralga exhibits enhanced CO₂ fixation compared to an otherwise identicalcontrol plant or alga grown under the same conditions.

29. The method of 28, wherein CO₂ fixation is enhanced in the range offrom about 10% to about 50% compared to that of an otherwise identicalcontrol plant or alga grown under the same conditions.

Another aspect of the present invention provides for a transgenic plantor alga made by the method of any one of 18-29.

Yet another aspect of the present invention provides for a biomass orother product from a plant or alga, selected from among starches, oils,fatty acids, lipids, cellulose or other carbohydrates, alcohols, sugars,nutraceuticals, pharmaceuticals, fragrance and flavoring compounds, andorganic acids, made by the method of any one of 18-29.

In addition to the various embodiments listed above, in the Examplesbelow, and in the claims, this disclosure further variously encompassesthe presently disclosed and claimed CCM protein combinations in furthercombinations with the genes and proteins focusing on engineeringpathways that are directly involved in facilitating CO₂ transport intoleaf chloroplasts, disclosed and claimed in the inventors' previousapplication PCT International Publication WO 2012/125737. The presentdisclosure encompasses any combination of genes disclosed herein withany combination of genes disclosed in WO 2012/125737 and in Tables D1-D9to improve carbon concentrating systems (CCMs) in plants and algae.

Table D1 represents different classes of α-CAs found in mammals.

Table D2-D4 represents representative species, Gene bank accessionnumbers, and amino acid sequences for various species of suitable CAgenes.

Table D5 represents the codon optimized DNA sequence for chloroplastexpression in Chlamydomonas reinhardtii. In Table D5, the underlinessequences represent restriction sites, and bases changed to optimizechloroplast expression are listed in lower case. Table D6 provides abreakdown of the number and type of each codon optimized.

Representative species and Gene bank accession numbers for variousspecies of bicarbonate transporter are listed below in Tables D8-D9.

Further scope of the applicability of the presently disclosedembodiments will become apparent from the detailed description anddrawing(s) provided below. However, it should be understood that thedetailed description and specific examples, while indicating preferredembodiments of this disclosure, are given by way of illustration onlysince various changes and modifications within the spirit and scope ofthese embodiments will become apparent to those skilled in the art fromthis detailed description.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The disclosure can be more fully understood form the following detaileddescription and the accompanying Sequence Listing, which form a part ofthis application.

The sequence descriptions summarize the Sequence Listing attachedhereto. The Sequence Listing contains standard symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. § 1.822.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentdisclosure will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawing(s), allof which are given by way of illustration only, and are not limitativeof the presently disclosed embodiments, in which:

FIG. 1. Model of the Chlamydomonas CCM showing the localization ofinorganic carbon transporters (HLA3, LCIA) and carbonic anhydrase (CAH:CAH1, CAH3, and CAH6) [5]), and Rubisco. LCIB is an essential proteinfor CCM in Chlamydomonas. It's exact function is unknown.

FIG. 2.(A-B) Growth phenotypes of VVT and HLA3 transgenic (T3)Arabidopsis initially grown on MS media (plus nitrate, NO3). (B) MSmedia (plus ammonium (NH4)+ and sucrose) or in soil (ammonium only). Xindicates plants died. Numbers refer to plant lines.

FIG. 3.(A-B) Growth phenotypes of VVT and HCA-II transgenic (T1)Arabidopsis 4 weeks after germination. (B) Growth phenotype of VVTArabidopsis (Col-0, left) and the BCA transgenic (T3) (right).

FIG. 4. Photosynthetic assimilation rate of CO₂ in three transgeniclines (P1, P5, P6) of Arabidopsis expressing BCA (bacterial carbonicanhydrase) measured using a LICOR 6400 gas analyzer. These lines showed˜30% increase in their photosynthetic efficiency when compared to WTArabidopsis (Col.-0).

FIG. 5.(A-C) Growth phenotypes of WT and LCIA transgenic (T1)Arabidopsis plants four weeks after germination. (B) Four-week-old WT(left 4 plants) and independent transgenic Camelina (right 4 plants)expressing LCIA. (C) CO₂-dependent photosynthetic rates of WT and LCIAtransgenic Camelina.

FIG. 6. Phenotype of HLA3 transgenics grown on nitrate. Energy chargeand reductive potential of WT and HLA3 transgenic Arabidopsis.Adenylate, nucleotide cofactors, and inorganic phosphate levels measuredas nmole/gFW for plants grown on nitrate. Values are averages±SE.

FIG. 7. Photosynthetically active radiation in proteorhodopsin relativeto plant-based chlorophyll [49].

FIG. 8. Plasmid pB110-CAB-PGR5-NOS (Example 1).

FIG. 9. Plasmid pB110-HLA3-pgr5-dsred (Example 1).

FIG. 10. Plasmid pBI 121-CAB1-Tp-NgCAf2-dsred (Example 1).

FIG. 11 illustrates light response curves of Camelina BCA lines.

FIG. 12 illustrates expression of LCIA in Camelina vs WT.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

The following detailed description is provided to aid those skilled inthe art in practicing the various embodiments of the present disclosuredescribed herein, including all the methods, uses, compositions, etc.,described herein. Even so, the following detailed description should notbe construed to unduly limit the present disclosure, as modificationsand variations in the embodiments herein discussed may be made by thoseof ordinary skill in the art without departing from the spirit or scopeof the present discoveries.

The present disclosure is explained in greater detail below. Thisdisclosure is not intended to be a detailed catalog of all the differentways in which embodiments of this disclosure can be implemented, or allthe features that can be added to the instant embodiments. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which variations and additions do not depart fromthe scope of the instant disclosure. Hence, the following specificationis intended to illustrate some particular embodiments of the disclosure,and not to exhaustively specify all permutations, combinations, andvariations thereof.

Any feature, or combination of features, described herein is(are)included within the scope of the present disclosure, provided that thefeatures included in any such combination are not mutually inconsistentas will be apparent from the context, this specification, and theknowledge of one of ordinary skill in the art. Additional advantages andaspects of the present disclosure are apparent in the following detaileddescription and claims.

The contents of all publications, patent applications, patents, andother references mentioned herein are incorporated by reference hereinin their entirety. In case of conflict, the present specification,including explanations of terms, will control.

Definitions

The following definitions are provided to aid the reader inunderstanding the various aspects of the present disclosure. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by those of ordinary skill inthe art to which the disclosure pertains.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth. Similarly, the word “or” is intended to include “and” unless thecontext clearly indicates otherwise. Hence “comprising A or B” meansincluding A, or B, or A and B. Furthermore, the use of the term“including”, as well as other related forms, such as “includes” and“included”, is not limiting.

The term “about” as used herein is a flexible word with a meaningsimilar to “approximately” or “nearly”. The term “about” indicates thatexactitude is not claimed, but rather a contemplated variation. Thus, asused herein, the term “about” means within 1 or 2 standard deviationsfrom the specifically recited value, or ±a range of up to 20%, up to15%, up to 10%, up to 5%, or up to 4%, 3%, 2%, or 1% compared to thespecifically recited value.

The term “comprising” as used in a claim herein is open-ended, and meansthat the claim must have all the features specifically recited therein,but that there is no bar on additional features that are not recitedbeing present as well. The term “comprising” leaves the claim open forthe inclusion of unspecified ingredients even in major amounts. The term“consisting essentially of” in a claim means that the inventionnecessarily includes the listed ingredients, and is open to unlistedingredients that do not materially affect the basic and novel propertiesof the invention. A “consisting essentially of” claim occupies a middleground between closed claims that are written in a closed “consistingof” format and fully open claims that are drafted in a “comprising’format”. These terms can be used interchangeably herein if, and when,this may become necessary. Furthermore, the use of the term “including”,as well as other related forms, such as “includes” and “included”, isnot limiting.

“BCA” refers to bacterial carbonic anhydrase.

“CCMs” and the like refer to carbon concentrating systems.

“CET” refers to cyclic electron transfer.

“LET” refers to linear electron transfer.

“WT” refers to wild-type.

“Cyclic electron transfer modulator protein” refers to any proteinnatural or synthetic that improves the separation of charge across thethylakoid membrane resulting in improved photophosphorylation with theproduction of chemical energy. Examples of such modulators are the PGR5and PRGL1 reductases, however improved proteins in the electrontransport chain such as cytochromes, ATPases, ferredoxin-NADP reductase,NAD(P)H-plastoquinone reductase, and the like are also CET modulatorproteins.

Unless otherwise stated, nucleic acid sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. Nucleic acid sequences may be provided as DNA or as RNA, asspecified; disclosure of one necessarily defines the other, as is knownto one of ordinary skill in the art and is understood as included inembodiments where it would be appropriate. Nucleotides may be referredto by their commonly accepted single-letter codes. Unless otherwiseindicated, amino acid sequences are written left to right in amino tocarboxyl orientation, respectively. Amino acids may be referred toherein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUM Biochemical NomenclatureCommission. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription purposes and are not to be unduly limiting.

Regarding disclosed ranges, the endpoints of all ranges directed to thesame component or property are inclusive and independently combinable(e.g., ranges of “up to about 25 wt. %, or, more specifically, about 5wt. % to about 20 wt. %,” is inclusive of the endpoints and allintermediate values of the ranges of “about 5 wt. % to about 25 wt. %,”etc.). Numeric ranges recited with the specification are inclusive ofthe numbers defining the range and include each integer within thedefined range.

As used herein, “altering level of production” or “altering level ofexpression” means changing, either by increasing or decreasing, thelevel of production or expression of a nucleic acid sequence or an aminoacid sequence (for example a polypeptide, an siRNA, a miRNA, an mRNA, agene), as compared to a control level of production or expression.

“Conservative amino acid substitutions”: It is well known that certainamino acids can be substituted for other amino acids in a proteinstructure without appreciable loss of biochemical or biologicalactivity. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid sequence substitutions can be made in a protein sequence,and, of course, its underlying DNA coding sequence, and neverthelessobtain a protein with like properties. Thus, various changes can be madein the amino acid sequences disclosed herein, or in the correspondingDNA sequences that encode these amino acid sequences, withoutappreciable loss of their biological utility or activity.

Proteins and peptides biologically functionally equivalent to theproteins and peptides disclosed herein include amino acid sequencescontaining conservative amino acid changes in the fundamental amino acidsequence. In such amino acid sequences, one or more amino acids in thefundamental sequence can be substituted, for example, with another aminoacid(s), the charge and polarity of which is similar to that of thenative amino acid, i.e., a conservative amino acid substitution,resulting in a silent change.

It should be noted that there are a number of different classificationsystems in the art that have been developed to describe theinterchangeability of amino acids for one another within peptides,polypeptides, and proteins. The following discussion is merelyillustrative of some of these systems, and the present disclosureencompasses any of the “conservative” amino acid changes that would beapparent to one of ordinary skill in the art of peptide, polypeptide,and protein chemistry from any of these different systems.

As disclosed in U.S. Pat. No. 5,599,686, certain amino acids in abiologically active peptide, polypeptide, or protein can be replaced byother homologous, isosteric, and/or isoelectronic amino acids, whereinthe biological activity of the original molecule is conserved in themodified peptide, polypeptide, or protein. The following list of aminoacid replacements is meant to be illustrative and is not limiting:

Original Replacement Amino Acid Amino Acid(s) Ala Gly Arg Lys, ornithineAsn Gln Asp Glu Glu Asp Gln Asn Gly Ala Ile Val, Leu, Met, Nle(norleucine) Leu Ile, Val, Met, Nle Lys Arg Met Leu, Ile, Nle, Val PheTyr, Trp Ser Thr Thr Ser Trp Phe, Tyr Tyr Phe, Trp Val Leu, Ile, Met,Nle

In another system, substitutes for an amino acid within a fundamentalsequence can be selected from other members of the class to which thenaturally occurring amino acid belongs. Amino acids can be divided intothe following four groups: (1) acidic amino acids; (2) basic aminoacids; (3) neutral polar amino acids; and (4) neutral non-polar aminoacids. Representative amino acids within these various groups include,but are not limited to: (1) acidic (negatively charged) amino acids suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cysteine, cystine, tyrosine,asparagine. and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within a fundamental peptide,polypeptide, or protein sequence can be made by substituting one aminoacid within one of these groups with another amino acid within the samegroup.

Some of the other systems for classifying conservative amino acidinterchangeability in peptides, polypeptides, and proteins applicable tothe sequences of the present disclosure include, for example, thefollowing:

Functionally defining common properties between individual amino acidsby analyzing the normalized frequencies of amino acid changes betweencorresponding proteins of homologous organisms (Schulz, G. E. and R. H.Schirmer (1979) Principles of Protein Structure (Springer Advanced Textsin Chemistry), Springer-Verlag). According to such analyses, groups ofamino acids can be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on overall protein structure;

Making amino acid changes based on the hydropathic index of amino acidsas described by Kyte and Doolittle (1982) J. Mol. Biol. 157(1):105-32.Certain amino acids can be substituted by other amino acids having asimilar hydropathic index or score and still result in a protein withsimilar biological activity, i.e., still obtain a biologicalfunctionally equivalent protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within +2 ispreferred, those that are within +1 are particularly preferred, andthose within +0.5 are even more particularly preferred;

Substitution of like amino acids on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 states that the greatest local average hydrophilicityof a protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein. As detailedin this patent, the following hydrophilicity values have been assignedto amino acid residues: arginine (+3.0); lysine (+3.0); aspartate(+3.0.+0.1); glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2);glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5.+0.1);alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4). Betts and Russell ((2003),“Amino Acid Properties and Consequences of Substitutions”,Bioinformatics for Geneticists, Michael R. Barnes and Ian C. Gray, Eds.,John Wiley & Sons, Ltd, Chapter 14, pp. 289-316) review the nature ofmutations and the properties of amino acids in a variety of differentprotein contexts with the purpose of aiding in anticipating andinterpreting the effect that a particular amino acid change will have onprotein structure and function. The authors point out that features ofproteins relevant to considering amino acid mutations include cellularenvironments, three-dimensional structure, and evolution, as well as theclassifications of amino acids based on evolutionary, chemical, andstructural principles, and the role for amino acids of different classesin protein structure and function in different contexts. The authorsnote that classification of amino acids into categories such as thoseshown in FIG. 14.3 of their review, which involves commonphysico-chemical properties, size, affinity for water (polar andnon-polar; negative or positive charge), aromaticity and aliphaticity,hydrogen-bonding ability, propensity for sharply turning regions, etc.,makes it clear that reliance on simple classifications can be dangerous,and suggests that alternative amino acids could be engineered into aprotein at each position. Criteria for interpreting how a particularmutation might affect protein structure and function are summarized insection 14.7 of this review, and include first inquiring about theprotein, and then about the particular amino acid substitutioncontemplated.

Biologically/enzymatically functional equivalents of the proteins andpeptides disclosed herein can have 10 or fewer conservative amino acidchanges, more preferably seven or fewer conservative amino acid changes,and most preferably five or fewer conservative amino acid changes, i.e.,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conservative amino acid changes. Theencoding nucleotide sequence (e.g., gene, plasmid DNA, cDNA,codon-optimized DNA, or other synthetic DNA) will thus havecorresponding base substitutions, permitting it to code for thebiologically functionally equivalent form of protein or peptide. Due tothe degeneracy of the genetic code, i.e., the existence of more than onecodon for most of the amino acids naturally occurring in proteins, otherDNA (and RNA) sequences that contain essentially the same geneticinformation as these nucleic acids, and which encode the same amino acidsequence as that encoded by these nucleic acids, can be used in themethods disclosed herein. This principle applies as well to any of theother nucleotide sequences disclosed herein.

“Control” or “control level” means the level of a molecule, such as apolypeptide or nucleic acid, normally found in nature under a certaincondition and/or in a specific genetic background. In certainembodiments, a control level of a molecule can be measured in a cell orspecimen that has not been subjected, either directly or indirectly, toa treatment. A control level is also referred to as a wildtype or abasal level. These terms are understood by those of ordinary skill inthe art. A control plant, i.e. a plant that does not contain arecombinant DNA that confers (for instance) an enhanced trait in atransgenic plant, is used as a baseline for comparison to identify anenhanced trait in the transgenic plant. A suitable control plant may bea non-transgenic plant of the parental line used to generate atransgenic plant. A control plant may in some cases be a transgenicplant line that comprises an empty vector or marker gene, but does notcontain the recombinant DNA, or does not contain all of the recombinantDNAs, in the test plant.

The terms “enhance”, “enhanced”, “increase”, or “increased” refer to astatistically significant increase. For the avoidance of doubt, theseterms generally refer to about a 5% increase in a given parameter orvalue, about a 10% increase, about a 15% increase, about a 20% increase,about a 25% increase, about a 30% increase, about a 35% increase, abouta 40% increase, about a 45% increase, about a 50% increase, about a 55%increase, about a 60% increase, about a 65% increase, about 70%increase, about a 75% increase, about an 80% increase, about an 85%increase, about a 90% increase, about a 95% increase, about a 100%increase, or more over the control value. These terms also encompassranges consisting of any lower indicated value to any higher indicatedvalue, for example “from about 5% to about 50%”, etc.

“Expression” or “expressing” refers to production of a functionalproduct, such as, the generation of an RNA transcript from an introducedconstruct, an endogenous DNA sequence, or a stably incorporatedheterologous DNA sequence. A nucleotide encoding sequence may compriseintervening sequence (e.g., introns) or may lack such interveningnon-translated sequences (e.g., as in cDNA). Expressed genes includethose that are transcribed into mRNA and then translated into proteinand those that are transcribed into RNA but not translated (for example,siRNA, transfer RNA, and ribosomal RNA). The term may also refer to apolypeptide produced from an mRNA generated from any of the above DNAprecursors. Thus, expression of a nucleic acid fragment, such as a geneor a promoter region of a gene, may refer to transcription of thenucleic acid fragment (e.g., transcription resulting in mRNA or otherfunctional RNA) and/or translation of RNA into a precursor or matureprotein (polypeptide), or both.

An “expression cassette” refers to a nucleic acid construct, which whenintroduced into a host cell, results in transcription and/or translationof a RNA or polypeptide, respectively.

The term “genome” as it applies to a plant cells encompasses not onlychromosomal DNA found within the nucleus, but organelle DNA found withinsubcellular components (e.g., mitochondrial, plastid) of the cell. Asused herein, the term “genome” refers to the nuclear genome unlessindicated otherwise. However, expression in a plastid genome, e.g., achloroplast genome, or targeting to a plastid genome such as achloroplast via the use of a plastid targeting sequence, is alsoencompassed by the present disclosure.

The term “heterologous” refers to a nucleic acid fragment or proteinthat is foreign to its surroundings. In the context of a nucleic acidfragment, this is typically accomplished by introducing such fragment,derived from one source, into a different host. Heterologous nucleicacid fragments, such as coding sequences that have been inserted into ahost organism, are not normally found in the genetic complement of thehost organism. As used herein, the term “heterologous” also refers to anucleic acid fragment derived from the same organism, but which islocated in a different, e.g., non-native, location within the genome ofthis organism. Thus, the organism can have more than the usual number ofcopy(ies) of such fragment located in its(their) normal position withinthe genome and in addition, in the case of plant cells, within differentgenomes within a cell, for example in the nuclear genome and within aplastid or mitochondrial genome as well. A nucleic acid fragment that isheterologous with respect to an organism into which it has been insertedor transferred is sometimes referred to as a “transgene.”

A “heterologous” PGRL1 protein or CAB transit peptide protein-encodingnucleotide sequence, etc., can be one or more additional copies of anendogenous PGRL1 protein or CAB transit peptide protein-encodingnucleotide sequence, or a nucleotide sequence from another plant orother source. PGRL1 is a putative ferredoxin-plastoquinone reductaseinvolved in photosynthetic cyclic electron flow. Furthermore, these canbe genomic or non-genomic nucleotide sequences. Non-genomic nucleotidesequences encoding such proteins and peptides include, by way ofnon-limiting examples, mRNA; synthetically produced DNA including, forexample, cDNA and codon-optimized sequences for efficient expression indifferent transgenic plants algae reflecting the pattern of codon usagein such plants; nucleotide sequences encoding the same proteins orpeptides, but which are degenerate in accordance with the degeneracy ofthe genetic code; which contain conservative amino acid substitutionsthat do not adversely affect their activity, etc., as known by those ofordinary skill in the art.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention can be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences, or homologs. The term “homologous” refers tothe relationship between two nucleic acid sequence and/or proteins thatpossess a “common evolutionary origin”, including nucleic acids and/orproteins from superfamilies (e.g., the immunoglobulin superfamily) inthe same species of animal, as well as homologous nucleic acids and/orproteins from different species of animal (for example, myosin lightchain polypeptide, etc.; see Reeck et al., (1987) Cell, 50:667). Suchproteins (and their encoding nucleic acids) may have sequence homology,as reflected by sequence similarity, whether in terms of percentidentity or by the presence of specific residues or motifs and conservedpositions. The methods disclosed herein contemplate the use of thepresently disclosed nucleic and protein sequences, as well as sequenceshaving sequence identity and/or similarity, and similar function.

“Host cell” means a cell which contains a vector and supports thereplication and/or expression of the vector. Host cells may beprokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Alternatively, the host cells aremonocotyledonous or dicotyledonous plant cells.

The term “introduced” means providing a nucleic acid (e.g., anexpression construct) or protein into a cell. “Introduced” includesreference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell, and includes reference to the transient provision ofa nucleic acid or protein to the cell. “Introduced” includes referenceto stable or transient transformation methods, as well as sexuallycrossing. Thus, “introduced” in the context of inserting a nucleic acidfragment (e.g., a recombinant DNA construct/expression construct) into acell, can mean “transfection” or “transformation” or “transduction”, andincludes reference to the incorporation of a nucleic acid fragment intoa eukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid, or mitochondrial DNA), converted into an autonomous replicon,or transiently expressed (e.g., transfected mRNA).

The term “isolated” refers to a material such as a nucleic acidmolecule, polypeptide, or small molecule, that has been separated fromthe environment from which it was obtained. It can also mean alteredfrom the natural state. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Thus, apolypeptide or polynucleotide produced and/or contained within arecombinant host cell is considered isolated. Also intended as “isolatedpolypeptides” or “isolated nucleic acid molecules”, etc., arepolypeptides or nucleic acid molecules that have been purified,partially or substantially, from a recombinant host cell or from anative source.

As used herein, “nucleic acid” or “nucleotide sequence” means apolynucleotide (or oligonucleotide), including single or double-strandedpolymers of deoxyribonucleotide or ribonucleotide bases, and unlessotherwise indicated, encompasses naturally occurring and syntheticnucleotide analogues having the essential nature of natural nucleotidesin that they hybridize to complementary single-stranded nucleic acids ina manner similar to naturally occurring nucleotides. Nucleic acids mayalso include fragments and modified nucleotide sequences. Nucleic acidsdisclosed herein can either be naturally occurring, for example genomicnucleic acids, or isolated, purified, non-genomic nucleic acids,including synthetically produced nucleic acid sequences such as thosemade by solid phase chemical oligonucleotide synthesis, enzymaticsynthesis, or by recombinant methods, including for example, cDNA,codon-optimized sequences for efficient expression in differenttransgenic plants reflecting the pattern of codon usage in such plants,nucleotide sequences that differ from the nucleotide sequences disclosedherein due to the degeneracy of the genetic code but that still encodethe protein(s) of interest disclosed herein, nucleotide sequencesencoding the presently disclosed protein(s) comprising conservative (ornon-conservative) amino acid substitutions that do not adversely affecttheir normal activity, PCR-amplified nucleotide sequences, and othernon-genomic forms of nucleotide sequences familiar to those of ordinaryskill in the art.

The protein-encoding nucleotide sequences, and promoter nucleotidesequences used to drive their expression, disclosed herein can begenomic or non-genomic nucleotide sequences. Non-genomic nucleotideprotein-encoding sequences and promoters include, for example,naturally-occurring mRNA, synthetically produced mRNA,naturally-occurring DNA, or synthetically produced DNA. Syntheticnucleotide sequences can be produced by means well known in the art,including by chemical or enzymatic synthesis of oligonucleotides, andinclude, for example, cDNA, codon-optimized sequences for efficientexpression in different transgenic plants and algae reflecting thepattern of codon usage in such organisms, variants containingconservative (or non-conservative) amino acid substitutions that do notadversely affect their normal activity, PCR-amplified nucleotidesequences, etc.

“A PGRL1 protein”, “a PGR5 protein”, “a HLA3 protein”, “a CAB transitpeptide”, “a PGR5 transit peptide”, or any other protein or peptidepresently broadly disclosed and utilized in any of the CCM methods andplants and algae disclosed herein refers to a protein or peptideexhibiting enzymatic/functional activity similar or identical to theenzymatic/functional activity of the specifically named protein orpeptide. Enzymatic/functional activities of the proteins and peptidesdisclosed herein are described below. “Similar” enzymatic/functionalactivity of a protein or peptide can be in the range of from about 75%to about 125% or more of the enzymatic/functional activity of thespecifically named protein or peptide when equal amounts of bothproteins or peptides are assayed, tested, or expressed as describedbelow under identical conditions, and can therefore be satisfactorilysubstituted for the specifically named proteins or peptides in thepresent enhanced CCM methods and transgenic plants and algae.

“Nucleic acid construct” or “construct” refers to an isolatedpolynucleotide which can be introduced into a host cell. This constructmay comprise any combination of deoxyribonucleotides, ribonucleotides,and/or modified nucleotides. This construct may comprise an expressioncassette that can be introduced into and expressed in a host cell.

“Operably linked” refers to a functional arrangement of elements. Afirst nucleic acid sequence is operably linked with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter effects the transcription or expression of the coding sequence.The control elements need not be contiguous with the coding sequence, solong as they function to direct the expression thereof. Thus, forexample, intervening untranslated yet transcribed sequences can bepresent between a promoter and the coding sequence and the promoter canstill be considered “operably linked” to the coding sequence.

The terms “plant” or “plants” that can be used in the present methodsbroadly include the classes of higher and lower plants amenable totransformation techniques, including angiosperms (monocotyledonous anddicotyledonous plants), gymnosperms, ferns, and unicellular andmulticellular algae. The term “plant” also includes plants which havebeen modified by breeding, mutagenesis, or genetic engineering(transgenic and non-transgenic plants). It includes plants of a varietyof ploidy levels, including aneuploid, polyploid, diploid, haploid, andhemizygous. The plant may be in any form including suspension cultures,embryos, meristematic regions, callus tissue, gametophytes, sporophytes,pollen, microspores, whole plants, shoot vegetative organs/structures(e.g. leaves, stems and tubers), roots, flowers and floralorgans/structures, seed (including embryo, endosperm, and seed coat) andfruit, plant tissue (e.g. vascular tissue, ground tissue, and the like)and cells, and progeny of same.

Embodiments of the present disclosure also include parts of plants oralgae, which can be selected from among a protoplast, a cell, a tissue,an organ, a cutting, an explant, a reproductive tissue, a vegetativetissue, biomass, an inflorescence, a flower, a sepal, a petal, a pistil,a stigma, a style, an ovary, an ovule, an embryo, a receptacle, a seed,a fruit, a stamen, a filament, an anther, a male or female gametophyte,a pollen grain, a meristem, a terminal bud, an axillary bud, a leaf, astem, a root, a tuberous root, a rhizome, a tuber, a stolon, a corm, abulb, an offset, a cell of said plant in culture, a tissue of said plantin culture, an organ of said plant in culture, a callus, propagationmaterials, germplasm, cuttings, divisions, and propagations.

Other embodiments include progeny or derivatives of transgenic plantsand algae disclosed herein selected, for example, from among clones,hybrids, samples, seeds, and harvested material. Progeny can beasexually or sexually produced by methods well known in the art.

Useful C3 and C4 Plants

Plants to which the methods disclosed herein can be advantageouslyapplied include both C3 and C4 plants, including “food crop” and“oilseed” plants, as well as algae.

Food Crop Plants

The term “food crop plant” refers to plants that are either directlyedible, or which produce edible products, and that are customarily usedto feed humans either directly, or indirectly through animals.Non-limiting examples of such plants include:

1. Cereal crops: wheat, rice, maize (corn), barley, oats, sorghum, rye,and millet;

2. Protein crops: peanuts, chickpeas, lentils, kidney beans, soybeans,lima beans;

3. Roots and tubers: potatoes, sweet potatoes, and cassavas;

4. Oil crops: soybeans, corn, canola, peanuts, palm, coconuts,safflower, cottonseed, sunflower, flax, olive, and safflower;

5. Sugar crops: sugar cane and sugar beets;

6. Fruit crops: bananas, oranges, apples, pears, breadfruit, pineapples,and cherries;

7. Vegetable crops and tubers: tomatoes, lettuce, carrots, melons,asparagus, etc.

8. Nuts: cashews, peanuts, walnuts, pistachio nuts, almonds;

9. Forage and turf grasses;

10. Forage legumes: alfalfa, clover;

11. Drug crops: coffee, cocoa, kola nut, poppy;

12. Spice and flavoring crops: vanilla, sage, thyme, anise, saffron,menthol, peppermint, spearmint, coriander.

In certain embodiments of this disclosure, the food crop plants aresoybean, canola, tomato, potato, cassava, wheat, rice, oats, lettuce,broccoli, beets, sugar beets, beans, peas, kale, strawberry, and peanut.

“Oilseed Plants”, “Oil Crop Plants”, “Biofuels Crops”, “Energy Crops”

The terms “oilseed plant” or “oil crop plant”, and the like, to whichthe present methods and compositions can also be applied, refer toplants that produce seeds or fruit with oil content in the range of fromabout 1 to 2%, e.g., wheat, to about 20%, e.g., soybeans, to over 40%,e.g., sunflowers and rapeseed (canola). These include major and minoroil crops, as well as wild plant species which are used, or are beinginvestigated and/or developed, as sources of biofuels due to theirsignificant oil production and accumulation.

Exemplary oil seed or oil crop plants useful in practicing the methodsdisclosed herein include, but are not limited to, plants of the generaBrassica (e.g., rapeseed/canola (Brassica napus; Brassica carinata;Brassica nigra; Brassica oleracea), Camelina, Miscanthus, and Jatropha;Jojoba (Simmondsia chinensis), coconut; cotton; peanut; rice; safflower;sesame; soybean; mustard; wheat; flax (linseed); sunflower; olive; corn;palm; palm kernel; sugarcane; castor bean; switchgrass; Boragoofficinalis; Echium plantagineum; Cuphea hookeriana; Cuphea pulcherrima;Cuphea lanceolata; Ricinus communis; Coriandrum sativum; Crepis alpina;Vernonia galamensis; Momordica charantia; and Crambe abyssinica.

A non-limiting example of a tuber that accumulates significant amountsof reserve lipids is the tuber of Cyperus esculentus (chufa ortigernuts), which has been proposed as an oil crop for biofuelproduction. In the case of chufa, use of a constitutive ortuber-specific promoter would be useful in the methods disclosed herein.

Useful Algae

Algae useful in practicing various methods of the present disclosureinclude members of the following divisions: Chlorophyta andHeterokontophyta.

In certain embodiments, useful algae include members of the followingclasses: Chlorophyceae, Bacillariophyceae, Eustigmatophyceae, andChrysophyceae. In certain embodiments, useful algae include members ofthe following genera: Nannochloropsis, Chlorella, Dunaliella,Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora,and Ochromonas. In one embodiment, members of the genus Chlorella arepreferred.

Some algal species of particular interest include, without limitation:Bacillariophyceae strains, Chlorophyceae, Cyanophyceae, Xanthophyceae,Chrysophyceae, Chlorella, Crypthecodinium, Schizocytrium,Nannochloropsis, Ulkenia, Dunaliella, Cyclotella, Navicula, Nitzschia,Cyclotella, Phaeodactylum, and Thaustochytrid.

Non-limiting examples of algae species that can be used with the methodsof the present disclosure include, for example, Achnanthes orientalis,Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphoracoffeiformis var. linea, Amphora coffeiformis var. punctata, Amphoracoffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphoradelicatissima, Amphora delicatissima var. capitata, Amphora sp.,Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekeloviahooglandii, Borodinella sp., Botryococcus braunii, Botryococcussudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria,Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var.subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorellaanitrata, Chlorella antarctica, Chlorella aureoviridis, ChlorellaCandida, Chlorella capsulate, Chlorella desiccate, Chlorellaellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var.vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorellainfusionum var. actophila, Chlorella infusionum var. auxenophila,Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis,Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var.lutescens, Chlorella miniata, Chlorella minutissima, Chlorellamutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva,Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides,Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorellaregularis var. minima, Chlorella regularis var. umbricata, Chlorellareisiglii, Chlorella saccharophila, Chlorella saccharophila var.ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana,Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorellavanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorellavulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorellavulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia,Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella,Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris,Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonassp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp.,Chlamydomonas moewusii Chlamydomonas reinhardtii Chlamydomonas sp.Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliellagranulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva,Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliellaterricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliellatertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp.,Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp.,Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonassp., Isochrysis aff. galbana, Isochrysis galbana, Lepocinclis,Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp.,Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Naviculaacceptata, Navicula biskanterae, Navicula pseudotenelloides, Naviculapelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp.,Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschiaclosterium, Nitzschia communis, Nitzschia dissipata, Nitzschiafrustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschiaintermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusillaelliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular,Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla,Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoriasubbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp.,Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp.,Pleurochrysis carterae, Pleurochrysis dentate, Pleurochrysis sp.,Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis,Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica,Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte,Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis,Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta,Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica,Thalassiosira weissflogii, and Viridiella fridericiana.

In certain embodiments of this disclosure, the algae are species ofChlorella, Nannochloropsis, and Chlamydomonas listed above.

Exemplary food crop plant include wheat, rice, maize (corn), barley,oats, sorghum, rye, and millet; peanuts, chickpeas, lentils, kidneybeans, soybeans, lima beans; potatoes, sweet potatoes, and cassavas;soybeans, corn, canola, peanuts, palm, coconuts, safflower, cottonseed,sunflower, flax, olive, and safflower; sugar cane and sugar beets;bananas, oranges, apples, pears, breadfruit, pineapples, and cherries;tomatoes, lettuce, carrots, melons, strawberry, asparagus, broccoli,peas, kale, cashews, peanuts, walnuts, pistachio nuts, almonds; forageand turf grasses; alfalfa, clover; coffee, cocoa, kola nut, poppy;vanilla, sage, thyme, anise, saffron, menthol, peppermint, spearmint andcoriander and preferably wheat, rice and canola.

The terms “peptide”, “polypeptide”, and “protein” are used to refer topolymers of amino acid residues. These terms are specifically intendedto cover naturally occurring biomolecules, as well as those that arerecombinantly or synthetically produced, for example by solid phasesynthesis.

The term “promoter” or “regulatory element” refers to a region ornucleic acid sequence located upstream or downstream from the start oftranscription and which is involved in recognition and binding of RNApolymerase and/or other proteins to initiate transcription of RNA.Promoters need not be of plant or algal origin. For example, promotersderived from plant viruses, such as the CaMV35S promoter, or from otherorganisms, can be used in variations of the embodiments discussedherein. Promoters useful in the present methods include, for example,constitutive, strong, weak, tissue-specific, cell-type specific,seed-specific, inducible, repressible, and developmentally regulatedpromoters.

A skilled person appreciates that a promoter sequence can be modified toprovide for a range of expression levels of an operably linkedheterologous nucleic acid molecule. Less than the entire promoter regioncan be utilized and the ability to drive expression retained. However,it is recognized that expression levels of mRNA can be decreased withdeletions of portions of the promoter sequence. Thus, the promoter canbe modified to be a weak or strong promoter. A promoter is classified asstrong or weak according to its affinity for RNA polymerase (and/orsigma factor); this is related to how closely the promoter sequenceresembles the ideal consensus sequence for the polymerase. Generally, by“weak promoter” is intended a promoter that drives expression of acoding sequence at a low level. By “low level” is intended levels ofabout 1/10,000 transcripts to about 1/100,000 transcripts to about1/500,000 transcripts. Conversely, a strong promoter drives expressionof a coding sequence at a high level, or at about 1/10 transcripts toabout 1/100 transcripts to about 1/1,000 transcripts. The promoter ofchoice is preferably excised from its source by restriction enzymes, butcan alternatively be PCR-amplified using primers that carry appropriateterminal restriction sites. It should be understood that the foregoinggroups of promoters are non-limiting, and that one skilled in the artcould employ other promoters that are not explicitly cited herein.

The term “purified” refers to material such as a nucleic acid, aprotein, or a small molecule, which is substantially or essentially freefrom components which normally accompany or interact with the materialas found in its naturally occurring environment, and/or which mayoptionally comprise material not found within the purified material'snatural environment. The latter may occur when the material of interestis expressed or synthesized in a non-native environment. Nucleic acidsand proteins that have been isolated include nucleic acids and proteinspurified by standard purification methods. The term also encompassesnucleic acids and proteins prepared by recombinant expression in a hostcell as well as chemically synthesized nucleic acids.

“Recombinant” refers to a nucleotide sequence, peptide, polypeptide, orprotein, expression of which is engineered or manipulated using standardrecombinant methodology. This term applies to both the methods and theresulting products. As used herein, a “recombinant construct”,“expression construct”, “chimeric construct”, “construct” and“recombinant expression cassette” are used interchangeably herein.

As used herein, the phrase “sequence identity” or “sequence similarity”is the similarity between two (or more) nucleic acid sequences, or two(or more) amino acid sequences. Sequence identity is frequently measuredas the percent of identical nucleotide or amino acid residues atcorresponding positions in two or more sequences when the sequences arealigned to maximize sequence matching, i.e., taking into account gapsand insertions.

One of ordinary skill in the art will appreciate that sequence identityranges are provided for guidance only. It is entirely possible thatnucleic acid sequences that do not show a high degree of sequenceidentity can nevertheless encode amino acid sequences having similarfunctional activity. It is understood that changes in nucleic acidsequence can be made using the degeneracy of the genetic code to producemultiple nucleic acid molecules that all encode substantially the sameprotein. Means for making this adjustment are well-known to those ofskill in the art. When percentage of sequence identity is used inreference to amino acid sequences it is recognized that residuepositions which are not identical often differ by conservative aminoacid substitutions, where amino acid residues are substituted for otheramino acid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. Where sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences which differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well-known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

Sequence identity (or similarity) can be readily calculated by knownmethods, including but not limited to those described in: ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determineidentity are designed to give the largest match between the sequencestested. Moreover, methods to determine identity are codified in publiclyavailable computer programs. Optimal alignment of sequences forcomparison can be conducted, for example, by the local homologyalgorithm of Smith & Waterman, by the homology alignment algorithms, bythe search for similarity method or, by computerized implementations ofthese algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG WisconsinPackage, available from Accelrys, Inc., San Diego, Calif., United Statesof America), or by visual inspection. See generally, (Altschul, S. F. etal., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. AcidsRes. 25: 3389-3402 (1997)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in (Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;& Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length Win the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold. These initial neighborhood word hits act as seedsfor initiating searches to find longer HSPs containing them. The wordhits are then extended in both directions along each sequence for as faras the cumulative alignment score can be increased. Cumulative scoresare calculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5877 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P (N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins can be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats, or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17: 149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17: 191-201 (1993)) low-complexity filters can beemployed alone or in combination.

The constructs and methods disclosed herein encompass nucleic acid andprotein sequences having sequence identity/sequence similarity at leastabout 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,100% to those specifically and/or sequences having the same or similarfunction for example if a protein or nucleic acid is identified with atransit peptide and the transit peptide is cleaved leaving the proteinsequence without the transit peptide then the sequence identity/sequencesimilarity is compared to the protein with and/or without the transitpeptide.

A “transgenic” organism, such as a transgenic plant, is a host organismthat has been stably or transiently genetically engineered to containone or more heterologous nucleic acid fragments, including nucleotidecoding sequences, expression cassettes, vectors, etc. Introduction ofheterologous nucleic acids into a host cell to create a transgenic cellis not limited to any particular mode of delivery, and includes, forexample, microinjection, floral dip, adsorption, electroporation, vacuuminfiltration, particle gun bombardment, whiskers-mediatedtransformation, liposome-mediated delivery, Agrobacterium-mediatedtransfer, the use of viral and retroviral vectors, etc., as is wellknown to those skilled in the art.

Conventional techniques of molecular biology, recombinant DNAtechnology, microbiology, and chemistry useful in practicing the methodsof the present disclosure are described, for example, in Green andSambrook (2012) Molecular Cloning: A Laboratory Manual, Fourth Edition,Cold Spring Harbor Laboratory Press; Ausubel et al. (2003 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.; Amberg et al. (2005) Methods in Yeast Genetics: A ColdSpring Harbor Laboratory Course Manual, 2005 Edition, Cold Spring HarborLaboratory Press; Roe et al. (1996) DNA Isolation and Sequencing:Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D.McGee (1990) In Situ Hybridization: Principles and Practice; OxfordUniversity Press; M. J. Gait (Editor) (1984) Oligonucleotide Synthesis:A Practical Approach, IRL Press; D. M. J. Lilley and J. E. Dahlberg(1992) Methods in Enzymology: DNA Structure Part A: Synthesis andPhysical Analysis of DNA, Academic Press; and Lab Ref: A Handbook ofRecipes, Reagents, and Other Reference Tools for Use at the Bench,Edited by Jane Roskams and Linda Rodgers (2002) Cold Spring HarborLaboratory Press; Burgess and Deutscher (2009) Guide to ProteinPurification, Second Edition (Methods in Enzymology, Vol. 463), AcademicPress. Note also U.S. Pat. Nos. 8,178,339; 8,119,365; 8,043,842;8,039,243; 7,303,906; 6,989,265; US20120219994A1; and EP1483367B1. Theentire contents of each of these texts and patent documents are hereinincorporated by reference.

Preliminary Results: Transgenic Plants Expressing Algal CCM Genes

Previously, reconstitution of a functional inorganic CCM in C3 plants tosuppress photo-respiration and enhance photosynthesis was proposed. InWO 2012/125737, it was hypothesized that expression of a minimum ofthree algal CCM proteins would be sufficient to elevate internal plastidCO₂ concentrations high enough to suppress photorespiration. These threealgal CCM genes included the Chlamydomonas plasma membrane-localized andATP-dependent bicarbonate transporter, HLA3; the chloroplast envelopelocalized bicarbonate anion transporter, LCIA; and a chloroplaststromal-localized carbonic anhydrase (HCA-II) to accelerate conversionof bicarbonate into CO₂. These genes have individually been shown to beimportant to the CCM in prior studies ([3-5]). To test this hypothesis,we generated multiple independent transgenic Arabidopsis and Camelinaplants expressing each CCM gene as a single gene construct, as well as astacked 3-gene construct. The expression of each gene was controlled bythe light-regulated Cab1 gene promoter [6].

The results of phenotypic analyses of Arabidopsis and Camelina plantstransformed with the single CCM gene constructs were as follows:

HLA3 Arabidopsis transgenics varied in their phenotypes, but generallyhad reduced growth phenotypes relative to wild-type (WT) plants (FIG.6). When the same plasmid was used to transform Camelina, no viableseeds were recovered from any transformation event after multipleattempts, indicating that HLA3 expression was likely toxic to Camelina.

With respect to carbonic anhydrase (CA) transgenics, we expressed ahuman carbonic anhydrase-2 (HCA2 (SEQ ID NO:17)) or a bacterialNeisseria gonorrhoeae carbonic anhydrase (BCA SEQ ID NO: 4)) in thechloroplast stroma [7]. We choose these CAs because each has a turnovernumber (Kcat=106 sec-1) that is approximately 10× faster thanplant/algal Cas. In both Arabidopsis and Camelina, we observedphenotypes that were either similar to WT (HCA2) or substantially larger(BCA) than WT plants (FIG. 3B).

Transgenic Arabidopsis plants expressing the LCIA gene weresubstantially impaired in growth (FIG. 5A). In contrast, Camelina LCIAtransgenics grew better than WT, had up to 25% higher photosyntheticrates at ambient CO₂ concentrations, and had reduced CO₂ compensationpoints (FIG. 5B).

The fact that expression of individual CCM genes impaired growth in C3plants suggested that additional traits may need to be expressed orsilenced to achieve optimal photosynthetic performance.

To determine if we could reconstitute a fully functional CCM complex inC3 plants, we transformed Arabidopsis and Camelina with a triple-geneCCM construct in which the expression of the HLA3, CA, and LCIA geneswas driven by the green-tissue specific Cab1 promoter. In bothArabidopsis and Camelina there was either a substantial impairment ingrowth, or the plants did not survive (results not shown).

Thus, co-expression of the HLA3 gene with any other CCM gene(s) impairedgrowth even in plants in which expression of the other CCM genes, e.g.,LCIA in Camelina, or BCA in Arabidopsis, enhanced growth. These resultsindicated that HLA3 expression was problematic.

Since the HLA3 protein catalyzes active bicarbonate transport and is thefirst-dedicated step in the engineered CCM, we re-focused our efforts ontrying to determine why HLA3 expression was toxic to plants and how tomitigate its effects. We considered two possible hypotheses for HLA3toxicity: 1) expression of the HLA3 ABC-transporter increases ATP demand(1 ATP/COO for photosynthesis by 25% and depletes cytoplasmic ATP levels[3-5,8] and 2) elevated bicarbonate levels in HLA3 transgenic plantsnegatively impact cytoplasmic pH levels. With respect to the latterhypothesis, it is noteworthy that unlike cyanobacteria, plants haverobust cytoplasmic CA activity, potentially mitigating the effects ofelevated bicarbonate levels on cytoplasmic pH.

The Role of ATP Demand and Cyclic Electron Transfer Activity in CCMs

In contrast to air-grown algae (4 ATP/2 NADPH/CO₂) and C4 plants (5ATP/2 NADPH/CO₂) which have increased ATP demands for photosynthesis, C3plants (3 ATP/2 NADPH/CO₂) have limited capacity to generate additionalATP for each electron transferred [8-10]. Increasing ATP demand by 25%per carbon fixed in HLA3 transgenic plants, therefore, could depletecytoplasmic ATP levels as well as alter the redox state of the cell[8,10]. One mechanism to increase ATP synthesis for each light-drivenelectron transferred is by cyclic electron transfer (CET) activity.Light-driven CET is catalyzed by photosystem I (PSI) mediated chargeseparation leading to the reduction of ferredoxin (fd) and the PGR5protein. The PGR5 protein reduces and protonates plastoquinone (PQ).PQH2 is then oxidized by the cytochrome b6f complex (Cyt b6f). Protonsreleased from the oxidation of PQH2 drive ATP synthesis. The electrontransfer cycle is completed by the reduction of plastocyanin (PC) by Cytb6f, which in turn is oxidized by the PSI primary donor P700+.Significantly, molecular studies have demonstrated that genes encodingproteins functional in CET are substantially overexpressed (4-10×) in C4plants and air-grown algae relative to related C3 species or high CO₂grown algae [9,11-17]. These CET genes include: the Proton GradientRegulation Genes PGR5 and PGRL1, and certain members of the Fd andferredoxin NADP reductase (FNR) gene families [8-15]: Accession Nos.:PGR5:NM_126585; PGRL1: NM_179091; Fd: AtFd1: At1g10960; AtFd2:At1g60950;FNR: LFNR1:At5g66190; LFRN2: At1g20020) [15]. The sequence for the PRG5protein with the transit peptide amino acid sequence underlined isprovided as

(SEQ ID NO: 1) MAAASISAIG CNQTLIGTSF YGGWGSSISG EDYQTMLSKTVAPPQQARVS RKAIRAVPMMKNVNEGKGLF APLVVVTRNLVGKKRFNQLR GKAIALHSQV ITEFCKSIGA DAKQRQGLIRAKKNGERLG FL.The transit peptide is cleaved to produce the functional PGR5 protein.

To test the hypothesis that ATP depletion in HLA3 transgenics resultedin growth impairment, we compared the phenotypes of VVT and HLA3transgenics grown on nitrate which would require more linear electrontransport (LET) to facilitate the reduction of nitrate. Significantly,the additional ATP produced by LET is not required for conversion ofnitrate to ammonium and thus total ATP levels are expected to increase.In contrast, plants grown on ammonium do not require additional LET.Finally, we also grew transgenics on ammonium with sucrose which wouldpresumably provide additional ATP via respiration [15,17]. Wehypothesized that growth on nitrate or ammonium with sucrose wouldprovide additional ATP that could potentially drive HLA3 activity.

As shown in FIG. 2B, none of the Arabidopsis HLA3 transgenics (4independent lines) grew in the presence of ammonium, but all HLA3 lineswere rescued when grown on ammonium with sucrose. Furthermore, plantsgrown on ammonium plus sucrose were phenotypically similar to VVT (FIG.2B). In contrast, all HLA3 plants grown on nitrate survived, but somelines (#9, #20) had substantially impaired growth phenotypes. Identicalresults were observed for the germination and growth of VVT and HLA3transgenic seeds on MS media agar plates using either nitrate (HLA3transgenics survived) or ammonium (HLA3 transgenics died) as the solenitrogen source (results not shown). Based on these observations, wepropose that increased ATP synthesis associated with nitrate-driven LETand/or sucrose metabolism reduces the depletion of cytoplasmic ATPlevels in HLA3 transgenics and rescues them.

This interpretation was corroborated by comparative metabolite analysesof leaf energy charge (EC) status (ATP), inorganic phosphate levels, andleaf reductive potential (RP) of VVT and HLA3 transgenic Arabidopsisgrown on nitrate. As shown in FIG. 6, HLA3 transgenics grown on nitratehad reduced EC and RP ratios relative to WT. Energy charge is defined as([ATP]+½[ADP])/([ATP]+[ADP]+[AMP]). The reduction potential is ameasurement of the capacity of the system to gain or lose electrons.

Significantly, inorganic phosphate levels were two-fold higher in HLA3line #20, while the NADH level was two-fold lower than WT.

These results are consistent with the hypothesis that HLA3 expressionplaces increased ATP demand on plants. This increased ATP demand in HLA3transgenics may be met in part via NAD(P)H oxidation via themalate/oxaloacetate redox shunt between the mitochondria andchloroplasts [16].

LCIA Phenotype Depends on Plant Species

As previously indicated, LCIA expression in transgenic Arabidopsisresulted in plants with severely depressed growth phenotypes (FIG. 5A).In contrast, transgenic Camelina expressing LCIA had increased growthrates as well as higher CO₂-dependent photosynthetic rates relative toWT (FIG. 5B). We propose that the substantially greater carbonsink-strength of Camelina relative to Arabidopsis accounts for theenhanced growth phenotype observed in Camelina LCIA plants. In supportof this hypothesis, we observed that Camelina LCIA transgenics hadhigher CO₂-dependent rates of photosynthesis and lower CO₂ compensationpoints (40 vs. 53 ppm CO₂) than WT plants indicative of facilitatedinorganic carbon uptake by LCIA (FIG. 5C).

Overview: Enhancing photosynthetic carbon fixation by increasing ATPproduction and limiting CO₂ diffusion out of artificial CCM lines;Strategies for facilitating CET and ATP synthesis in C3 plants

Prior attempts to subvert the limitations of photosynthesis have focusedon engineering RuBisCO throughput and specificity [35] by introductionof engineered and non-native forms of the enzyme [36], throughalterations in the regenerative capacity of the Calvin cycle [37,38] orby engineering photorespiratory bypasses [39]. These studies producedmixed results, thus advocating for a more comprehensive systems-levelapproach to enhance and/or redirect photosynthetic carbon flux.

As evidenced by our prior work described above, we postulate that boththe carbon assimilatory steps and the light-based generation of ATP andNAPDH must be considered to develop a competent CCM with significantlyimproved photosynthetic capacity. To demonstrate proof of concept, anArabidopsis line that contains a functional CCM that includes mechanismsto adjust ATP levels to meet transporter demand will be generated.

Enhancing CET and ATP Synthesis to Support HLA3-Dependent BicarbonateUptake

To exploit the expression of an algal CCM in C3 plants requires that wemeet the additional energy demands required to actively transportinorganic carbon. As previously discussed in the section entitled “Therole of ATP demand and cyclic electron transfer activity in CCMs”, C4plants and algae have robust CET activity, and overexpress a variety ofgenes involved in CET [13,16,40-45] compared to C3 plants.

Several strategies are identified in the following examples, to increaseATP synthesis to support HLA3-dependent bicarbonate transport. Severalof these strategies focus on elevating CET activity in C3 plants.Another approach involves the expression of a green photon-drivenbacterial proton pump in thylakoids to supplement proton-driven ATPsynthesis. Each approach is designed to complement existing CCM lines inArabidopsis, Camelina, and potato we have created, and are evaluatedbased upon measured adenylate levels, plant biomass production, andphotosynthetic measurements of carbon assimilation. The materials andmethods employed in the examples below are for illustrative purposesonly, and are not intended to limit the practice of the presentembodiments thereto. Any materials and methods similar or equivalent tothose described herein as would be apparent to one of ordinary skill inthe art can be used in the testing or practice of the presentembodiments, i.e., the materials, methods, and examples are illustrativeonly and not intended to be limiting.

Example 1: Enhancing CET Based on Overexpressing the Proton GradientRegulatory Proteins PGR5 and PGRL1 in C3 Plants

Enhancing CET is based on overexpressing the proton gradient regulatoryproteins PGR5 and/or PGRL1 which have previously been shown to beimportant to CET [37].

It has recently been demonstrated that the PGRL1 protein has antimycinA-sensitive (AA), ferredoxin-plastoquinone reductase (FQR) activity[46]. In Chlamydomonas, PGRL1 is part of the Cytb6f/PSI supercomplexwhich mediates CET. Significantly, PGRL1 forms homodimers as well asheterodimers with PGR5 via redox active cysteine residues. Underhigh-light conditions, thioredoxinred reduces PGRL1 dimers present ingrana stacks, increasing the abundance of PGRL1 monomers and enhancingCET [47]. Mutational studies have shown that the PGR5 protein isrequired for Fd oxidation and PGRL1 reduction, but not for PQ reduction.In addition, it has been shown that PGRL1/PGR5 heterodimers are moreactive in CET than PGRL1 monomers. In C4 plants PGR5 and PGRL1expression levels are elevated (4×) relative to C3 plants [9].Similarly, PGR5 expression is up-regulated in air-grown Chlamydomonas(active CCM and HLA3 activity) relative to high CO₂ (low CCM) growncells [16,43]. Significantly, overexpression of PGRL1 and PGR5 has alsobeen shown to increase AA-sensitive CET in transgenic Arabidopsis [48].One embodiment of the present invention provides for an overexpressionof PGRL1 gene (SEQ ID NO:106) and PGR5 gene with chloroplast targetingsequence (SEQ ID NO:2) with HLA3 gene (SEQ ID NO:12) or with HLA3 gene(SEQ ID NO:12) and LCIA gene (SEQ ID NO:16) and BCA gene codon optimizedfor expression in Arabidopsis (SEQ ID NO:4) to yield substantiallyincreased photosynthetic rates, particularly in plants with enhancedsink strength (Camelina and potato for example). Co-expression of thePGR5 gene (SEQ ID NO:2) along with the HLA3 gene (SEQ ID NO:12) inCamelina rescued the HLA3 gene and it was no longer lethal. Theseresults indicate that the PGR5 gene is enabling the production ofsufficient ATP to meet the demands of the HLA3 gene product.

HLA3 (SEQ ID NO:12) and PGR5 (SEQ ID NO:2) are introduced as a doubleconstruct into Arabidopsis or Camelina, by Agrobacterium-mediated Tiplasmid transformation using, for example, plasmid pB110-HLA3-pgr5-dsred(FIG. 9). Since PGR5 protein (SEQ ID NO:1) is naturally targeted to thethylakoid membranes, no additional targeting sequences are introduced.Similarly, since HLA3 protein (SEQ ID NO:77) is naturally targeted tothe chloroplast envelope, no additional targeting sequences are added.HLA3 is codon optimized for plant expression.

In one embodiment, the expression of each protein is driven by the lightsensitive leaf-specific CAB1 promoter (SEQ ID NO:7) and Nos terminator(SEQ ID NO:9) (FIG. 9).

The BCA gene (AAW89307; SEQ ID NO:4), under the control of CAB1promoter, is introduced in to Arabidopsis by Agrobacterium-mediated Tiplasmid transformation by floral dip method using the construct shown inFIG. 10.

As a visual marker, the plasmid also includes a gene for expression offluorescent DsRed protein under the control of CVMV promoter and Nosterminator (FIG. 10).

Plants are transformed by vacuum infiltration method (Lu and Kang(February, 2008) Plant Cell Rep. 27(2):273-8), and will be screened forbiomass yield parameters (including plant weight, height, branching andseed yield) and photosynthetic efficiency measured as CO₂ absorptionwith the aid of a LiCor 6400 gas exchange analyzer.

The PGRL1 gene from Arabidopsis (NM_179091 SEQ ID NO:3) will besubcloned into pCambia1301-based binary plasmid under control of theCAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQ ID NO:9). Theplasmid will also carry a gene for hygromycin selection marker.Agrobacterium-mediated transformation takes place by the standard floraldip method followed by germination of seeds on hygromycin to select fortransformants. The expression of PRGL1 will be confirmed by RT-PCR, andthe resulting transgenic plant lines will be crossed with HLA3/PGR5plants and screened for biomass yield and photosynthesis rate (CO₂fixation).

Example 2: Determining if Fd1 Gene Overexpression can Support Algal CCMand Increased Photosynthetic Rates

It has recently been demonstrated that specific members of theferredoxin (Fd) gene family facilitate CET. Overexpression of peaferredoxin1 (Fd1) enhanced CET at the expense of LET in tobacco [16,40].

Therefore, another embodiment of the present invention providesenhancing ATP production and titrating the expression of the pea Fd1gene in the three model C3 plants with and without co-expression of theCCM genes to determine if Fd1 overexpression can support the algal CCMand increased photosynthetic rates. Earlier results demonstrated thatFd1 overexpression slightly impaired Linear Electron Transfer (LET),resulting in a stunted phenotype [40]. We expect that the additional ATPdemand in HLA3 transgenics, however, will mitigate these effects.

Fd1 gene (At1g10960) will be introduced by Agrobacterium-mediated Tiplasmid transformation. Fd1 gene will be subcloned intopCambia1301-based binary plasmid under control of CAB1 promoter (SEQ IDNO:7) and Nos terminator (SEQ ID NO:9). The plasmid will also carry agene for hygromycin selection as a marker. Agrobacterium-mediatedtransformation takes place by the standard floral dip method, followedby germination of seeds on hygromycin to select for transformants. Theexpression of FD1 (SEQ ID NO:93) will be confirmed by real time QPCR,and the resulting plant lines exhibiting different levels of FD1expression will be crossed with CCM-expressing plants and screened forbiomass yield and photosynthesis rate with the aid of a LiCor 6400CO₂-gas exchange analyzer.

Example 3: Overexpression of Unique Ferredoxin NADP Reductase (FNR) GeneFamily Members Associated with CET

Yet another embodiment is based on overexpression of unique ferredoxinNADP reductase (FNR) gene family members associated with CET. Leaf FNR(LFNR) catalyzes the reduction of Fd and is involved in both LET and CET[15]. It was recently demonstrated that there are three LFNR gene familymembers expressed in maize leaves: Accession Nos. BAA88236 (LFNR1),BAA88237 (LFNR2), and ACF85815 (LFNR3).

LFNR-1 was shown to be localized to thylakoid membranes and associatedwith Cytb6f complexes. LFNR2 was present in thylakoids and stromaassociated with Cytb6f complexes. LFNR3 was soluble and not associatedwith Cytb6f complexes.

Significantly, when plants were grown with nitrate instead of ammonium,expression of LFNR1 and LFNR2 was elevated but not that of LFNR3. Incontrast, studies using Arabidopsis LFNR1 knock out mutants demonstratedthat PGA-dependent oxygen evolution (which requires additional ATP) ismore negatively affected than is nitrate-dependent oxygen evolution (noadditional ATP demand), suggesting that LFNR1 may play a role inregulating CET [15]. However, this interpretation remains equivocal.

To determine if CET activity and HLA3 mediated inorganic carbon uptakecan be altered by differential expression of LFNR1, we will bothover-express (CAB1 promoter (SEQ ID NO:7)) and under-express (LFNR1RNAi) LFNR1 in transgenic Arabidopsis to determine the impact of alteredLFNR1 expression on functional CCM activity.

For overexpression of the LFNR1, the gene (At5g66190) will be introducedby Agrobacterium-mediated Ti plasmid transformation by floral dipping.The LFNR1gene will be subcloned into pCambia1301-based binary plasmidunder control of the CAB1 promoter (SEQ ID NO:7) and Nos terminator (SEQID NO:9). The plasmid will also carry a gene for hygromycin selection asa marker. The expression of LFNR1 will be confirmed by real time QPCR,the resulting plant lines will be crossed with CCM-expressing plants,and screened for biomass yield and photosynthesis rate with the aid of aLiCor 6400 CO₂-gas exchange analyzer.

For downregulaton of the LFNR1 levels, an RNAi construct containing apartial sequence of the LFNR1 (At5g66190 or BAA88236) and reversecomplementary sequence of LFNR1 will be subcloned into pCambia1301-basedbinary plasmid under control of the CAB1 promoter (SEQ ID NO:7) and Nosterminator (SEQ ID NO:9). The plasmid will also carry a gene forhygromycin selection as a marker. The reduced level of LFNR1 expressionwill be confirmed by real time QPCR.

The resulting lines will be crossed with CCM-expressing lines togenerate double mutants. Those mutants will be screened for biomassyield parameters (including plant weight, height, branching and seedyield) and photosynthetic efficiency measured as CO₂ absorption with theaid of a LiCor 6400 gas exchange analyzer.

Example 4: Facilitated Vectoral Proton Transport Using Proteorhodopsin(PR)

In yet another embodiment green photons, not absorbed by chlorophyll, todrive proton transport across thylakoids by expressing modified PR [49])will be employed to enhance ATP synthesis (FIG. 7).

PR is a seven-helix transmembrane-spanning protein similar tobacteriorhodopsin that contains retinal in its active site. Greenlight-driven cis-trans isomerization of retinal drives vectoral protontransfer across the membrane [50-55]. Significantly, it has beendemonstrated that a functional PR could be expressed in arespiration-impaired mutant of E. coli when supplemented with exogenousall-trans retinal [56]. More recently, hydrogen production was shown toincrease nearly two-fold in PR-expressing E. coli when cells wereexposed to increasing light intensities (70 to 130 μE), indicating thatPR can efficiently absorb light even at low intensities [57]. To thebest of our knowledge, retinal complementation of other rhodopsins hasnot been reported. Significantly, PR-expressing E. coli respiratorymutants generated sufficient proton-motive force to support ATPsynthesis levels, leading to enhanced cell viability and motility whentransgenics were exposed to sunlight as the only energy source.

These results suggest that targeting PR to the thylakoid membrane usingappropriate targeting sequences (e.g., nuclear-encoded, N-terminal,light harvesting complex signal sequences) and supplementation withexogenous retinal or retinal derived from β-carotene cleavage) coulddrive additional ATP synthesis. One concern is that the optical crosssection of retinal is small and light harvesting by PR is notsupplemented by antenna complexes. This constraint may be overcome inpart by overexpressing PR in thylakoids. Regardless, the additionalproton gradient necessary to support HLA3 activity is substantially lessthan that required to support overall CO₂ fixation. The best achievablePR expression levels will be determined empirically using different genepromoters, e.g., psaD (SEQ ID NO:10), rbcs (SEQ ID NO:11), and cab1 (SEQID NO:7), to drive its expression.

Generation of Improved PR and its Functional Reconstitution inChloroplasts

PR (AF279106), for example (SEQ ID NO:98), will be introduced intoArabidopsis, Camelina, and potato by Ti plasmid transformation andtargeted to the thylakoid membrane using the DNAJ transit peptide(At5g21430, SEQ ID NO: 22) or psbX stop-transfer trans-membrane domain(At2g06520 SEQ ID NO:23) fused to the C-terminus of PR [58], or transitpeptides from nuclear encoded chloroplast proteins such as CAB (SEQ IDNO:13), PGR5 (SEQ ID NO:14), and psaD (SEQ ID NO:15). Reconstitutionwith exogenous retinal will be carried out in a manner similar tostrategies described for E. coli, except that retinal will be painted onthe surface of the leaf [56] to demonstrate proof of concept. Retinalreconstitution will be followed by monitoring the absorption of thethylakoid membranes at 540 nm [59].

If exogenously applied retinal is not incorporated into PR, we willexpress low levels of a plant codon-optimized β-carotene monooxygenasefor example (SEQ ID NO:100) in plastids to cleave a small fraction ofβ-carotene to generate retinal. Non-limiting examples of β-carotenemonooxygenases that can be used include, for example, mouse, human,zebra fish, and rat enzymes (Accession Nos. AW044715, AK001592,AJ290390, and NM_053648, respectively). Alternatively, if β-carotenelevels are severely depleted, we will transiently express β-carotenemonooxygenase under the control of a transient inducible promoter suchas an ethanol inducible gene promoter. This is available as anEcoRl/Pstl fragment from Syngenta-Construct: pJL67-5S::AlcR/AlcA::GUS inpMLBART (Weigel World, Max Planck Institute for Developmental Biology,Tubingen, Germany) for periods of time sufficient to fully saturate PR[60,61]. Operation of a functional retinal photocycle in PR will beconfirmed by transient absorption spectroscopy [62].

Alternatively, promoters such as the green tissue/leaf-specificpromoters such as the CAB (At3g54890 SEQ ID NO:7) and rbcS (At5g38420SEQ ID NO:11) promoters can be used, for example see SEQ ID NO:5 for theBCA protein with a rbc-1a transit peptide. As the skilled person will bewell aware, various promoters may be used to promote the transcriptionof the nucleic acid of the invention, i.e. the nucleic acid which whentranscribed yields an RNA molecule that modulates the expression and/oractivity of a protein according to the invention. Such promoters includefor example constitutive promoters, inducible promoters (e.g. lightinducible promoters, stress-inducible promoters, drought-induciblepromoters, hormone-inducible promoters, chemical-inducible promoters,etc.), tissue-specific promoters, developmentally regulated promotersand the like.

Thus, a plant expressible promoter can be a constitutive promoter, i.e.a promoter capable of directing high levels of expression in most celltypes (in a spatio-temporal independent manner). Examples of plantexpressible constitutive promoters include promoters of bacterialorigin, such as the octopine synthase (OCS) and nopaline synthase (NOS)promoters from Agrobacterium, but also promoters of viral origin, suchas that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapsteret al., 1988, Mol. Gen. Genet. 212: 182-190) or 19S RNAs genes (Odell etal., 1985, Nature. 6; 313(6005):810-2; U.S. Pat. No. 5,352,605; WO84/02913; Benfey et al., 1989, EMBO J. 8:2195-2202), the enhanced 2×35Spromoter (Kay at al., 1987, Science 236:1299-1302; Datla et al. (1993),Plant Sci 94:139-149) promoters of the cassava vein mosaic virus (CsVMV;WO 97/48819, U.S. Pat. No. 7,053,205), 2×CsVMV (WO2004/053135) thecircovirus (AU 689 311) promoter, the sugarcane bacilliform badnavirus(ScBV) promoter (Samac et al., 2004, Transgenic Res. 13(4):349-61), thefigwort mosaic virus (FMV) promoter (Sanger et al., 1990, Plant MolBiol. 14(3):433-43), the subterranean clover virus promoter No 4 or No 7(WO 96/06932) and the enhanced 35S promoter as described in U.S. Pat.Nos. 5,164,316, 5,196,525, 5,322,938, 5,359,142 and 5,424,200. Among thepromoters of plant origin, mention will be made of the promoters of thepromoter of the Arabidopsis thaliana histone H4 gene (Chabouté et al.,1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol.29:637-649, U.S. Pat. No. 5,510,474) of Maize, Rice and sugarcane, theRice actin 1 promoter (Act-1, U.S. Pat. No. 5,641,876), the histonepromoters as described in EP 0 507 698 A1, the Maize alcoholdehydrogenase 1 promoter (Adh-1) (from the world wide web atpatentlens.net/daisy/promoters/242.html)).

A variety of plant gene promoters that regulate gene expression inresponse to environmental, hormonal, chemical, developmental signals,and in a tissue-active manner can be used for expression of a sequencein plants. Choice of a promoter is based largely on the phenotype ofinterest and is determined by such factors as tissue (e.g., seed, fruit,root, pollen, vascular tissue, flower, carpel, etc.), inducibility(e.g., in response to heat, cold, drought, light etc.), timing,developmental stage, and the like.

Promoters that can be used to practice this invention include those thatare green tissue specific such as the promoter of light harvestingcomplex protein 2 (Sakamoto et al. Plant Cell Physiology, 1991, 32(3):385-393) or the promoter of the cytosolic fructose-1, 6-bisphosphatasefrom rice (Si et al. Acta Botanica Sinica 45: 3(2003): 359-364).Alternative embodiments include light inducible promoters such aspromoters of the plant ribulose-biscarboxylase/oxygenase (Rubisco) smallsubunit promoter (U.S. Pat. No. 4,962,028; WO99/25842) from Zea mays andsunflower. Also the small subunit promoter from Chrysanthemum may beused, combined or not combined with the use of the respective terminator(Outchkourov et al., Planta, 216: 1003-1012, 2003).

Additional promoters that can be used to practice this invention arethose that elicit expression in response to stresses, such as the RD29promoters that are activated in response to drought, low temperature,salt stress, or exposure to ABA (Yamaguchi-Shinozaki et al., 2004, PlantCell, Vol. 6, 251-264; WO12/101118), but also promoters that are inducedin response to heat (e.g., see Ainley et al. (1993) Plant Mol. Biol. 22:13-23), light (e.g., the pea rbcS-3A promoter, Kuhlemeier et al. (1989)Plant Cell 1: 471-478, and the maize rbcS promoter, Schaffher and Sheen(1991) Plant Cell 3: 997-1012); wounding (e.g., wunl, Siebertz et al.(1989) Plant Cell 1: 961-968); pathogens (such as the PR-I promoterdescribed in Buchel et al. (1999) Plant Mol. Biol. 40: 387-396, and thePDF 1.2 promoter described in Manners et al. (1998) Plant Mol. Biol. 38:1071-1080), and chemicals such as methyl jasmonate or salicylic acid(e.g., see Gatz (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). In addition, the timing of the expression can be controlled byusing promoters such as those acting at senescence (e.g., see Gan andAmasino (1995) Science 270: 1986-1988); or late seed development (e.g.,see Odell et al. (1994) Plant Physiol. 106: 447-458).

Use may also be made of salt-inducible promoters such as thesalt-inducible NHX1 promoter of rice landrace Pokkali (PKN) (Jahan etal., 6^(th) International Rice Genetics symposium, 2009, poster abstractP4-37), the salt inducible promoter of the vacuolar H+-pyrophosphatasefrom Thellungiella halophila (TsVP1) (Sun et al., BMC Plant Biology2010, 10:90), the salt-inducible promoter of the Citrus sinensis geneencoding phospholipid hydroperoxide isoform gpx1 (Avsian-Kretchmer etal., Plant Physiology July 2004 vol. 135, p 1685-1696).

In alternative embodiments, tissue-specific and/or developmentalstage-specific promoters are used, e.g., promoter that can promotetranscription only within a certain time frame of developmental stagewithin that tissue. See, e.g., Blazquez (1998) Plant Cell 10:791-800,characterizing the Arabidopsis LEAFY gene promoter. See also Cardon(1997) Plant J 12:367-77, describing the transcription factor SPL3,which recognizes a conserved sequence motif in the promoter region ofthe A. thaliana floral meristem identity gene API; and Mandel (1995)Plant Molecular Biology, Vol. 29, pp 995-1004, describing the meristempromoter elF4. Tissue specific promoters which are active throughout thelife cycle of a particular tissue can be used. Other promoters that canbe used to express the nucleic acids of the invention include, aleaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285 1295,describing a leaf-specific promoter in maize); a tomato promoter activeduring fruit ripening, senescence and abscission of leaves, a guard-cellpreferential promoter e.g. as described in PCT/EP12/065608, and, to alesser extent, of flowers can be used (see, e.g., Blume (1997) Plant J.12:731 746); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBELI gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.Further tissue specific promoters that may be used according to theinvention include, promoters active in vascular tissue (e.g., see Ringliand Keller (1998) Plant Mol. Biol. 37: 977-988), carpels (e.g., see Ohlet al. (1990) Plant Cell 2. In alternative embodiments, plant promoterswhich are inducible upon exposure to plant hormones, such as auxins, areused to express the nucleic acids used to practice the invention. Forexample, the invention can use the auxin-response elements EI promoterfragment (AuxREs) in the soybean {Glycine max L.) (Liu (1997) PlantPhysiol. 115:397-407); the auxin-responsive Arabidopsis GST6 promoter(also responsive to salicylic acid and hydrogen peroxide) (Chen (1996)Plant J. 10: 955-966); the auxin-inducible parC promoter from tobacco(Sakai (1996) 37:906-913); a plant biotin response element (Streit(1997) Mol. Plant Microbe Interact. 10:933-937); and, the promoterresponsive to the stress hormone abscisic acid (ABA) (Sheen (1996)Science 274:1900-1902). Further hormone inducible promoters that may beused include auxin-inducible promoters (such as that described in vander Kop et al. (1999) Plant Mol. Biol. 39: 979-990 or Baumann et al.,(1999) Plant Cell 11: 323-334), cytokinin-inducible promoter (e.g., seeGuevara-Garcia (1998) Plant Mol. Biol. 38: 743-753), promotersresponsive to gibberellin (e.g., see Shi et al. (1998) Plant Mol. Biol.38: 1053-1060, Willmott et al. (1998) Plant Molec. Biol. 38: 817-825)and the like.

In alternative embodiments, nucleic acids used to practice the inventioncan also be operably linked to plant promoters which are inducible uponexposure to chemicals reagents which can be applied to the plant, suchas herbicides or antibiotics. For example, the maize In2-2 promoter,activated by benzenesulfonamide herbicide safeners, can be used (DeVeylder (1997) Plant Cell Physiol. 38:568-577); application of differentherbicide safeners induces distinct gene expression patterns, includingexpression in the root, hydathodes, and the shoot apical meristem.Coding sequence can be under the control of, e.g., atetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324). Usingchemically- {e.g., hormone- or pesticide) induced promoters, i.e.,promoter responsive to a chemical which can be applied to the transgenicplant in the field, expression of a polypeptide of the invention can beinduced at a particular stage of development of the plant. Use may alsobe made of the estrogen-inducible expression system as described in U.S.Pat. No. 6,784,340 and Zuo et al. (2000, Plant J. 24: 265-273) to drivethe expression of the nucleic acids used to practice the invention.

In alternative embodiments, a promoter may be used whose host range islimited to target plant species, such as corn, rice, barley, wheat,potato or other crops, inducible at any stage of development of thecrop.

In alternative embodiments, a tissue-specific plant promoter may driveexpression of operably linked sequences in tissues other than the targettissue. In alternative embodiments, a tissue-specific promoter thatdrives expression preferentially in the target tissue or cell type, butmay also lead to some expression in other tissues as well, is used.

According to the invention, use may also be made, in combination withthe promoter, of other regulatory sequences, which are located betweenthe promoter and the coding sequence, such as transcription activators(“enhancers”), for instance the translation activator of the tobaccomosaic virus (TMV) described in Application WO 87/07644, or of thetobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol.64: 1590-1597, for example.

Other regulatory sequences that enhance the expression of the nucleicacid of the invention may also be located within the chimeric gene. Oneexample of such regulatory sequences is introns. Introns are interveningsequences present in the pre-mRNA but absent in the mature RNA followingexcision by a precise splicing mechanism. The ability of natural intronsto enhance gene expression, a process referred to as intron-mediatedenhancement (IME), has been known in various organisms, includingmammals, insects, nematodes and plants (WO 07/098042, p 11-12). IME isgenerally described as a posttranscriptional mechanism leading toincreased gene expression by stabilization of the transcript. The intronis required to be positioned between the promoter and the codingsequence in the normal orientation. However, some introns have also beendescribed to affect translation, to function as promoters or as positionand orientation independent transcriptional enhancers (Chaubet-Gigot etal., 2001, Plant Mol Biol. 45(1):17-30, p 27-28).

Examples of genes containing such introns include the 5′ introns fromthe rice actin 1 gene (see U.S. Pat. No. 5,641,876), the rice actin 2gene, the maize sucrose synthase gene (Clancy and Hannah, 2002, PlantPhysiol. 130(2):918-29), the maize alcohol dehydrogenase-1 (Adh-1) andBronze-1 genes (Callis et al. 1987 Genes Dev. 1(10):1183-200;Mascarenhas et al. 1990, Plant Mol Biol. 15(6):913-20), the maize heatshock protein 70 gene (see U.S. Pat. No. 5,593,874), the maize shrunken1 gene, the light sensitive 1 gene of Solanum tuberosum, and the heatshock protein 70 gene of Petunia hybrida (see U.S. Pat. No. 5,659,122),the replacement histone H3 gene from alfalfa (Keleman et al. 2002Transgenic Res. 11(1):69-72) and either replacement histone H3 (histoneH3.3-like) gene of Arabidopsis thaliana (Chaubet-Gigot et al., 2001,Plant Mol Biol. 45(1):17-30).

Other suitable regulatory sequences include 5′ UTRs. As used herein, a5′ UTR, also referred to as a leader sequence, is a particular region ofa messenger RNA (mRNA) located between the transcription start site andthe start codon of the coding region. It is involved in mRNA stabilityand translation efficiency. For example, the 5′ untranslated leader of apetunia chlorophyll a/b binding protein gene downstream of the 35Stranscription start site can be utilized to augment steady-state levelsof reporter gene expression (Harpster et al., 1988, Mol Gen Genet.212(1):182-90). WO95/006742 describes the use of 5′ non-translatedleader sequences derived from genes coding for heat shock proteins toincrease transgene expression.

The chimeric gene may also comprise a 3′ end region, i.e. atranscription termination or polyadenylation sequence, operable in plantcells. As a transcription termination or polyadenylation sequence, usemay be made of any corresponding sequence of bacterial origin, such asfor example the nos terminator of Agrobacterium tumefaciens, of viralorigin, such as for example the CaMV 35S terminator, or of plant origin,such as for example a histone terminator as described in publishedPatent Application EP 0 633 317 A1. The polyadenylation region can bederived from the natural gene, from a variety of other plant genes, orfrom T-DNA. The 3′ end sequence to be added may be derived from, forexample, the nopaline synthase or octopine synthase genes, oralternatively from another plant gene, or less preferably from any othereukaryotic gene.

The expression and targeting of proteorhodopsin to the thylakoidmembranes will take advantage of the green energy spectrum that isinaccessible to chlorophyll. An increase in the amount of ATP isexpected under photosynthesis conditions, from proton gradient generatedboth by the photosystems and the proteorhodopsin pump. Under conditionsof inhibition of electron transfer through the photosystems, we shouldbe able to observe a steady rate of ATP synthesis well above the basalrate through the activity of the proteorhodopsin proton pump.

Under normal pH conditions, protons are pumped into the bacterialperiplasmic space by PR [50]. The photo-driven retinal cycle begins withphotoisomerization of all trans-retinal to 13-cis retinal. The resultingconformational change poises the system for transfer of a proton fromthe Schiff base (SB; pKa˜11) to the counter ion, Asp 97 (pKa˜7.5). Theproton is transferred to the lumen via a proton-conducting channel, andthe SB is reprotonated from the cytoplasm. The mechanism of protonrelease in PR is not as well understood as in bacteriorhodopsin (BR);however, the main events of the photocycle are expected to be similar tothose of BR. One potential challenge for pumping protons by PR inthylakoid membranes is the pH gradient-dependent reversibility of protontransfer by PR. At periplasmic pHs, <5.5, proton flow in PR is reversed,potentially depleting the proton gradient and impairing ATP synthesis.Thus, at the lumenal pH of thylakoids (4.5), reversed protontransduction via PR is possible. One of the critical residues involvedin reversible proton flow is Asp97, which acts as the proton acceptorfrom retinal. The pKa of Asp97 in PR is ˜7.5, while the pKa of itscounterpart in BR is ˜2.5. Due to the extremely low pKa of the counterion, BR is able to retain its forward pumping activity at pHs as low as3.5. The ability of PR to act as a proton pump in the thylakoid membranethus entails maintaining the pumping efficiency at low pH conditionsprevailing in the lumen. We propose that vectoral pumping of protonsinto the thylakoid lumen can be achieved by lowering the pKa of Asp97and/or by protecting the SB from the lumenal pH through rational,site-specific mutagenesis. The electrostatic environment around the SBin PR is presumably maintained by the counter ions, Asp97, Asp227(analogous to BR Asp212), Arg94 (analogous to BR Arg82) and His75. InBR, the low pKa of Asp85 is attributed to its strong hydrogen bondinginteractions with Thr89 and Arg82 [53,54]. Since, interactions thatreduce the pKa of Asp97 will promote proton-pumping activity at lowexternal pH, mutation of Met79 to a residue that can hydrogen bond toHis75 and Asp212, like Tyr or Thr, will be explored. These mutations areproposed by overlaying the structures of BR and PR, and identifyingresidues which are in a position to effect the desired behavior.Finally, the ability of a modified PR to work as an efficient H+ pump atacidic pHs will also entail shielding the SB from the extracellularenvironment. To this end, a L219E/T206S mutant will be generated,wherein E219 and S206 will form a Glu-Ser gate regulating vectoralproton transfer as occurs in BR.

To determine if any transgenes alter CET or ATP synthesis activity, wewill compare the dark reduction kinetics of the photosystem I primarydonor, P700+ in VVT and transgenic plants, with and withoutdibromothymoquinone (DBMIB), an inhibitor of Cytb6f-mediated CET. DarkP700+ reduction kinetics are expected to be faster in plants with moreactive CET. In addition, we will assess the amplitude of the After Glow(AG) thermoluminescence band (−40° C.) associated with CET activity[11,14,16,43,63]. Pool sizes of ATP will also be assessed in VVT andtransgenic plants by mass spectroscopy.

Referring now to FIG. 11, additional transgenic Camelina lines wereproduced that expressed the BCA gene (SEQ ID NO:4) in the chloroplaststroma. These lines were produced using the Agrobacterium-mediatedtransformation procedures as described previously. Three lines wereevaluated for their ability to accumulate biomass and provide improvedphotosynthetic rates. Wildtype Camelina and the BCA mutant lines werenot significantly different at lower light levels (0-400 umol/m²/s) intheir ability to assimilate carbon dioxide. However, as light intensityincreased the BCA transformants showed between 10 and 30% higheraccumulation of CO₂ at 2000 μmoles/m²/s than wildtype. The BCA line 9.2was the highest while lines BCA 4.1 and BCA 5.7 were both about 10%higher than wildtype. This improved ability to assimilate CO₂ wasreflected in two of the lines (BCA-5.7 and BCA-9.2) into increasedbiomass accumulation, with these lines having about 15% greater biomassaccumulation than wildtype. The BCA-4.1 line did not show improvedbiomass accumulation compared to control.

Referring now to FIG. 12, the ability of the chloroplast envelopedlocalized bicarbonate transporter bicarbonate transporter (LCIA) proteinto transport bicarbonate and improve the capture of inorganic carbon bytransgenic Camelina was determined following the method of Farquhar andcolleagues (1989). LCIA transgenic Camelina were produced using theAgrobacterium-mediated transformation processed described previously. ALCIA expressing mutant line (CAM-LCIA) was compared to wildtype Camelina(Cam-WT) for the observed discrimination of the stable isotope ¹³C. Thiscarbon isotope discrimination is expressed as the difference between the¹³C in the air and in a plant which has been previously exposed to¹³CO₂, the carbon isotope discrimination is symbolized by A andexpressed in parts per million (ppm) and is described by Farquhar andcolleagues (1989). In the LCIA transgenic lines, the observeddiscrimination by the plant was 20% less than that observed in thewildtype. This indicates that the insertion of LCIA provides the plantthe ability to better accumulate and retain inorganic carbon than thewildtype plant and shows decreased “leakiness” vs wildtype. Referencefor ¹³C discrimination: Carbon isotope discrimination andphotosynthesis, G. D. Farquhar, J. R. Ehlieringer and K. T. Hubick.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1989, 40, 503-537.

TABLE D1 Kcat/ Kcat Km Km Ki Subcellular Tissue I organ Isoenzyme (s−1)(mM) (M⁻¹s⁻¹) (nM) localization localization hCAI  2 × 10⁵ 4.0 5.0 × 10⁷250 cytosol E, GI hCAII  1.4 × 10^(6o) 9.3 1.5 × 10⁸ 12 cytosol E, eye,GI, BO, K, L, T, B hCAIII 1.0 × 10⁴ 33.3 3.0 × 10⁵ 2 × 10⁵ cytosol SM, AhCAIV 1.0 × 10⁶ 21.5 5.1 × 10⁷ 74 membrane K, L, P, B, C, H hCAVA 2.9 ×10⁵ 10.0 2.9 × 10⁷ 63 mitochondria Li hCAVB 9.5 × 10⁵ 9.7 9.8 × 10⁷ 54mitochondria H, SM, P, K, SC, GI hCAVI 3.4 × 10⁵ 6.9 4.9 × 10⁷ 11secreted G hCAVII 9.5 × 10⁵ 11.4 8.3 × 10¹ 2.5 cytosol CNS hCAVIIIcytosol CNS hCAIX 3.8 × 10⁵ 6.9 5.5 × 10⁷ 25 transmembrane TU, GI hCAXcytosol CNS hCAXI cytosol CNS hCAXII 4.2 × 10⁵ 12.0 3.5 × 10⁷ 5.7transmembrane R, I, RE, eye, TU hCAXIII 1.5 × 10⁵ 13.8 1.1 × 10⁷ 16cytosol K, B, L, GI, RE hCAXIV 3.1 × 10⁵ 7.9 3.9 × 10⁷ 41 transmembraneK, B, L hCAXV 4.7 × 10⁵ 14.2 3.3 × 10⁷ 72 membrane K H = Human; M =Mouse; hCAVIII, X, and XI are devoid of catalytic activity. E =Erthrocyes; GI = GI tract; BO = Bone osteoclasts; K = kidney, L = Lung;T = testis; B = brain; SM = skeletal muscle; A = Adipocytes; P =pancreas; C = colon; H = heart; Li = liver; SC = spinal cord; G =salivary and mammary gland; R = renal; I = intestinal; TU = tumors, RE =Reproductive

TABLE D2 Exemplary Type II Carbonic Anhydrases Accession OrganismSequence Number SEQ. ID. NO HumanMSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY NP-000058.1 SEQ. ID. NO. 19DPSLKPLSVS YDQATSLRIL NNGHAFNVEF DDSQDKAVLKGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QVLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK MacacaMSHHWGYGKH NGPEHWHKDF PIAKGQRQSP VDIDTHTAKY BAE91302.1 SEQ. ID. NO. 24fascicularis DPSLKPLSVS YDQATSLRIL NNGHSFNVEF DDSQDKAVIK (crab-eatingGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHL macaque)VHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMSKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK PanMSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY NP_001181853 SEQ. ID. NO. 25troglodytes DPSLKPLSVS YGQATSLRIL NNGHAFNVEF DDSQDKAVLKGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP HGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMLKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFK MacacaMSHHWGYGKH NGPEHWHKDF PIAKGQRQSP VDINTHTAKY NP_001182346 SEQ. ID. NO. 26mulatta DPSLKPLSVS YDQATSLRIL NNGHSFNVEF DDSQDKAVIKGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMSKFRKLNF NGEGEPEELM VDNWRPAQPL KNRQIKASFKPongo abelii MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002819286SEQ. ID. NO. 27 DPSLKPLSVC YDQATSLRIL NNGHSFNVEF DDSQDKAVLKGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KCADFTNFDP RGLLPASLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMLKFRKLNF NGEGEPEELM VDNWRPAQPL KKRQIKASFKCallithrix MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002759086SEQ. ID. NO. 28 jacchus DPSLKPLSVS YDQATSWRIL NNGHSFNVEF DDSQDKAVLKGGPLDGTYRL IQFHFHWGST DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAAQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLESVTWIV LKEPISVSSE QILKFRKLNF SGEGEPEELM VDNWRPAQPL KNRQIKASFKLemur catta MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDINTGAAKH ADD83028SEQ. ID. NO. 29 DPSLKPLSVY YEQATSRRIL NNGHSFNVEF DDSQDKAVLKGGPLDGTYRL IQFHFHWGSL DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKVG SAKPGLQKVVDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYLGSLTTPPLLECVTWIV LKEPISVSSE QMMKFRKLSF SGEGEPEELM VDNWRPAQPL KNRQIKASFKAiluropoda MAHHWGYGKH NGPEHWYKDF PIAKGQRQSP VDIDTKAAIH XP_002916939SEQ. ID. NO. 30 melanoleuca DPALKALCPT YEQAVSQRVI NNGHSFNVEF DDSQDNAVLKGGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKIG DARPGLQKVLDALDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMLKFRRLNF NKEGEPEELM VDNWRPAQPL HNRQINASFK EguusMSHHWGYGQH NGPKHWHKDF PIAKGQRQSP VDIDTKAAVH XP_001488540 SEQ. ID. NO. 31caballus DAALKPLAVH YEQATSRRIV NNGHSFNVEF DDSQDKAVLQGGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVVGVFLKVG GAKPGLQKVLDVLDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LREPISVSSE QLLKFRSLNF NAEGKPEDPM VDNWRPAQPL NSRQIRASFKCanis lupus MAHHWGYAKH NGPEHWHKDF PIAKGERQSP VDIDTKAAVH NP_001138642SEQ. ID. NO. 32 familiaris DPALKSLCPC YDQAVSQRII NNGHSFNVEF DDSQDKTVLKGGPLTGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYGEF GKAVQQPDGL AVLGIFLKIG GANPGLQKILDALDSIKTKG KSADFTNFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPISVSSE QMLKFRKLNF NKEGEPEELM MDNWRPAQPL HSRQINASFKOryctolagus MSHHWGYGKH NGPEHWHKDF PIANGERQSP IDIDTNAAKH NP_001182637SEQ. ID. NO. 33 cuniculus DPSLKPLRVC YEHPISRRII NNGHSFNVEF DDSHDKTVLKEGPLEGTYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHLVHWNTKYGDF GKAVKHPDGL AVLGIFLKIG SATPGLQKVVDTLSSIKTKG KSVDFTDFDP RGLLPESLDY WTYPGSLTTPPLLECVTWIV LKEPITVSSE QMLKFRNLNF NKEAEPEEPM VDNWRPTQPL KGRQVKASFVAiluropoda GPEHWYKDFP IAKGQRQSPV DIDTKAAIHD PALKALCPTY EFB24165SEQ. ID. NO. 34 melanoleuca EQAVSQRVIN NGHSFNVEFD DSQDNAVLKG GPLTGTYRLIQFHFHWGSSD GQGSEHTVDK KKYAAELHLV HWNTKYGDFGKAVQQPDGLA VLGIFLKIGD ARPGLQKVLD ALDSIKTKGKSADFTNFDPR GLLPESLDYW TYPGSLTTPP LLECVTWIVLKEPISVSSEQ MLKFRRLNFN KEGEPEELMV DNWRPAQPLH NRQINASFK Sus scrofaMSHHWGYDKH NGPEHWHKDF PIAKGDRQSP VDINTSTAVH XP_001927840.1SEQ. ID. NO. 35 DPALKPLSLC YEQATSQRIV NNGHSFNVEF DSSQDKGVLEGGPLAGTYRL IQFHFHWGSS DGQGSEHTVD KKKYAAELHLVHWNTKYKDF GEAAQQPDGL AVLGVFLKIG NAQPGLQKIVDVLDSIKTKG KSVEFTGFDP RDLLPGSLDY WTYPGSLTTPPLLESVTWIV LREPISVSSG QMMKFRTLNF NKEGEPEHPM VDNWRPTQPL KNRQIRASFQCallithrix MSHHWGYGKH NGPEHWHKDF PIAKGERQSP VDIDTHTAKY XP_002759087SEQ. ID. NO. 36 jacchus DPSLKPLSVS YDQATSWRIL NNGHSFNVEF DDSQDKAVLKGGPLDGTYRL IQLHLVHWNT KYGDFGKAAQ QPDGLAVLGIFLKVGSAKPG LQKVVDVLDS IKTKGKSADF TNFDPRGLLPESLDYWTYPG SLTTPPLLES VTWIVLKEPI SVSSEQILKFRKLNFSGEGE PEELMVDNWR PAQPLKNRQI KASFK MusMSHHWGYSKH NGPENWHKDF PIANGDRQSP VDIDTATAQH NP_033931 SEQ. ID. NO. 37musculus DPALQPLLIS YDKAASKSIV NNGHSFNVEF DDSQDNAVLKGGPLSDSYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHLVHWNTKYGDF GKAVQQPDGL AVLGIFLKIG PASQGLQKVLEALHSIKTKG KRAAFANFDP CSLLPGNLDY WTYPGSLTTPPLLECVTWIV LREPITVSSE QMSHFRTLNF NEEGDAEEAM VDNWRPAQPL KNRKIKASFKBos taurus MSHHWGYGKH NGPEHWHKDF PIANGERQSP VDIDTKAVVQ NP_848667SEQ. ID. NO. 38 DPALKPLALV YGEATSRRMV NNGHSFNVEY DDSQDKAVLKDGPLTGTYRL VQFHFHWGSS DDQGSEHTVD RKKYAAELHLVHWNTKYGDF GTAAQQPDGL AVVGVFLKVG DANPALQKVLDALDSIKTKG KSTDFPNFDP GSLLPNVLDY WTYPGSLTTPPLLESVTWIV LKEPISVSSQ QMLKFRTLNF NAEGEPELLM LANWRPAQPL KNRQVRGFPKOryctolagus GKHNGPEHWH KDFPIANGER QSPIDIDTNA AKHDPSLKPL AAA80531SEQ. ID. NO. 39 cuniculus RVCYEHPISR RIINNGHSFN VEFDDSHDKT VLKEGPLEGTYRLIQFHFHW GSSDGQGSEH TVNKKKYAAE LHLVHWNTKYGDFGKAVKHP DGLAVLGIFL KIGSATPGLQ KVVDTLSSIKTKGKSVDFTD FDPRGLLPES LDYWTYPGSL TTPPLLECVTWIVLKEPITV SSEQMLKFRN LNFNKEAEPE EP RattusMSHHWGYSKS NGPENWHKEF PIANGDRQSP VDIDTGTAQH NP062164 SEQ. ID. NO. 40norvegicus DPSLQPLLIC YDKVASKSIV NNGHSFNVEF DDSQDFAVLKEGPLSGSYRL IQFHFHWGSS DGQGSEHTVN KKKYAAELHLVHWNTKYGDF GKAVQHPDGL AVLGIFLKIG PASQGLQKITEALHSIKTKG KRAAFANFDP CSLLPGNLDY WTYPGSLTTPPLLECVTWIV LKEPITVSSE QMSHFRKLNF NSEGRAEELM VDNWRPAQPL KNRKIKASFK

TABLE D3 Exemplary Type VII Carbonic Anhydrases Accession OrganismSequence Number SEQ. ID. NO HumanMSLSITNNGH SVQVDFNDSD DRTVVTGGPL EGPYRLKQFH   SEQ. ID. NO. 41FHWGKKHDVG SEHTVDGKSF PSELHLVHWN AKKYSTFGEAASAPDGLAVV GVFLETGDEH PSMNRLTDAL YMVRFKGTKAQFSCFNPKCL LPASRHYWTY PGSLTTPPLS ESVTWIVLREPICISERQMG KFRSLLFTSE DDERIHMVNN FRPPQPLKGR VVKASFRA PongoMTGHHGWGYG QDDGPSHWHK LYPIAQGDRQ SPINIISSQA XP_002826555 SEQ. ID. NO. 42abelii VYSPSLQPLE LSYEACMSLS ITNNGHSVQV DFNDSDDRTVVTGGPLEGPY RLKQFHFHWG KKHDVGSEHT VDGKSFPSELHLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMNRLTDALYMVR FKGTKAQFSCFNPKSLLPAS RHYWTYPGSLTTPPLSESVT WIVLREPICI SERQMGKFRS LLFTSEDDER IHMVNNFRPP QPLKGRVVKA SFRAPan MEFGLSPELS PSRCFKRLLR GSERGRSRSP NERTEPTGQV XP_001143159.1SEQ. ID. NO. 43 troglodytes HGCGDGSGMT GHHGWGYGQD DGPSHWHKLY PIAQGDRQSPINIISSQAVY SPSLQPLELS YEACMSLSIT NNGHSVQVDFNDSDDRTVVT GGPLEGPYRL KQFHFHWGKK HDVGSEHTVDGKSFPSELHL VHWNAKKYST FGEAASAPDG LAVVGVFLETGDEHPSMNRL TDALYMVRFK GTKAQFSCFN PKCLLPASRHYWTYPGSLTT PPLSESVTWI VLREPICISE RQMRKFRSLLFTSEDDERIH MVNNFRPPQP LKGRVVKASF RA CallithrixMTGHHGWGYG QDDGPSHWHK LYPIAQGDRQ SPINIISSQA XP_002761099 SEQ. ID. NO. 44jacchus VYSPSLQPLE LSYEACMSLS ITNNGHSVQV DFNDSDDRTVVTGGPLEGPY RLKQFHFHWG KKHDVGSEHT VDGKSFPSELHLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMNRLTDALYMVR FKGTKAQFSC FNPKCLLPAS WHYWTYPGSLTTPPLSESVT WIVLREPICI SERQMGKFRS LLFTSEDDER VHMVNNFRPP QPLKGRVVKA SFRAAiluropoda GPSQWHKLYP IAQGDRQSPI NIVSSQAVYS PSLKPLELSY EFB15849SEQ. ID.NO. 45 melanoleuca EACISLSIAN NGHSVQVDFN DSDDRTVVTG GPLDGPYRLKQFHFHWGKKH SVGSEHTVDG KSFPSELHLV HWNAKKYSTFGEAASAPDGL AVVGVFLETG DEHPSMNRLT DALYMVRFKGTKAQFSCFNP KCLLPASRHY WTYPGSLTTP PLSESVTWIVLREPISISER QMEKFRSLLF TSEDDERIHM VNNFRPPQPL KGRVVKASFR A CanisMTGHHCWGYG QNDEIQASLS PSLSTPAGPS QWHKLYPIAQ XP_546892 SEQ. ID. NO. 46familiaris GDRQSPINIV SSQAVYSPSL KPLELSYEAC ISLSITNNGHSVQVDFNDSD DRTAVTGGPL DGPYRLKQLH FHWGKKHSVGSEHTVDGKSF PSELHLVHWN AKKYSTFGEA ASAPDGLAVVGIFLETGDEH PSMNRLTDAL YMVRFKGTKA QFSCFNPKCLLPASRHYWTY PGSLTTPPLS ESVTWIVLRE PISISERQMEKFRSLLFTSE EDERIHMVNN FRPPQPLKGR VVKASFRA Bos taurusMTGHHGWGYG QNDGPSHWHK LYPIAQGDRQ SPINIVSSQA XP_002694851 SEQ. ID. NO. 47VYSPSLKPLE ISYESCTSLS IANNGHSVQV DFNDSDDRTVVSGGPLDGPY RLKQFHFHWG KKHGVGSEHT VDGKSFPSELHLVHWNAKKY STFGEAASAP DGLAVVGVFL ETGDEHPSMNRLTDALYMVR FKGTKAQFSC FNPKCLLPAS RHYWTYPGSLTTPPLSESVT WIVLREPIRI SERQMEKFRS LLFTSEEDER IHMVNNFRPP QPLKGRVVKA SFRARattus MTVLWWPMLR EELMSKLRTG GPSNWHKLYP IAQGDRQSPI EDL87229SEQ. ID. NO. 48 norvegicus NIISSQAVYS PSLQPLELFY EACMSLSITN NGHSVQVDFNDSDDRTVVAG GPLEGPYRLK QLHFHWGKKR DVGSEHTVDGKSFPSELHLV HWNAKKYSTF GEAAAAPDGL AVVGIFLETGDEHPSMNRLT DALYMVRFKD TKAQFSCFNP KCLLPTSRHYWTYPGSLTTP PLSESVTWIV LREPIRISER QMEKFRSLLFTSEDDERIHM VNNFRPPQPL KGRVVKASFQ S OryctolagusMTGHHGWGYG QDDGGRPSHW HKLYPIAQGD RQSPINIVSS XP_002711604 SEQ. ID. NO. 49cuniculus QAVYSPGLQP LELSYEACTS LSIANNGHSV QVDFNDSDDRTVVTGGPLEG PYRLKQFHFH WGKRRDAGSE HTVDGKSFPSELHLVHWNAR KYSTFGEAAS APDGLAVVGV FLETGNEHPSMNRLTDALYM VRFKGTKAQF SCFNPKCLLP SSRHYWTYPGSLTTPPLSES VTWIVLREPI SISERQMEKF RSLLFTSEDD ERVHMVNNFR PPQPLRGRVV KASFRAMus GQDDGPSNWH KLYPIAQGDR QSPINIISSQ AVYSPSLQPL AAG16230.1SEQ. ID. NO. 50 musculus ELFYEACMSL SITNNGHSVQ VDFNDSDDRT VVSGGPLEGPYRLKQLHFHW GKKRDMGSEH TVDGKSFPSE LHLVHWNAKKYSTFGEAAAA PDGLAVVGVF LETGDEHPSM NRLTDALYMVRFKDTKAQFS CFNPKCLLPT SRHYWTYPGS LTTPPLSESVTWIVLREPIR ISERQMEKFR SLLFTSEDDE RIHMVDNFRP PQPLKGRVVK ASFQA MonodelphisMTGHHGWGYG QEDGPSEWHK LYPIAQGDRQ SPIDIVSSQA XP_001364411.1SEQ. ID. NO. 51 domestic VYDPTLKPLV LAYESCMSLS IANNGHSVMV EFDDVDDRTVVNGGPLDGPY RLKQFHFHWG KKHSLGSEHT VDGKSFSSELHLVHWNGKKY KTFAEAAAAP DGLAVVGIFL ETGDEHASMNRLTDALYMVR FKGTKAQFNS FNPKCLLPMN LSYWTYPGSLTTPPLSESVT WIVLKEPITI SEKQMEKFRS LLFTAEEDEK VRMVNNFRPP QPLKGRVVQA SFRSGallus MTGHHSWGYG QDDGPAEWHK SYPIAQGNRQ SPIDIISAKA XP_414152.1SEQ. ID. NO. 52 gallus VYDPKLMPLV ISYESCTSLN ISNNGHSVMV EFEDIDDKTVISGGPFESPF RLKQFHFHWG AKHSEGSEHT IDGKPFPCELHLVHWNAKKY ATFGEAAAAP DGLAVVGVFL EIGKEHANMNRLTDALYMVK FKGTKAQFRS FNPKCLLPLS LDYWTYLGSLTTPPLNESVI WVVLKEPISI SEKQLEKFRM LLFTSEEDQK VQMVNNFRPP QPLKGRTVRA SFKATaeniopygia MTGQHSWGYG QADGPSEWHK AYPIAQGNRQ SPIDIDSARA XP_002190292.1SEQ. ID. NO. 53 guttata VYDPSLQPLL ISYESCSSLS ISNTGHSVMV EFEDTDDRTAISGGPFQNPF RLKQFHFHWG TTHSQGSEHT IDGKPFPCELHLVHWNARKY TTFGEAAAAP DGLAVVGVFL EIGKEHASMNRLTDALYMVK FKGTKAQFRG FNPKCLLPLS LDYWTYLGSLTTPPLNESVT WIVLKEPIRI SVKQLEKFRM LLFTGEEDQR IQMANNFRPP QPLKGRIVRA SFKA

TABLE D4 Exemplary Type XIII Carbonic Anhydrases Accession OrganismSequence Number SEQ. ID. NO HumanMSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTKEVK NP_940986.1 SEQ. ID. NO. 54YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTENKSVLRGGPLTGSYR LRQVHLHWGS ADDHGSEHIV DGVSYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQKITDTLDSIKE KGKQTRFTNF DLLSLLPPSW DYWTYPGSLTVPPLLESVTW IVLKQPINIS SQQLAKFRSL LCTAEGEAAA FLVSNHRPPQ PLKGRKVRAS FH PanMSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTKEVK XP_001169377.1SEQ. ID. NO. 55 troglodytes YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTENKSVLRGGPLTGSYR LRQFHLHWGS ADDHGSEHIV DGVSYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQKITDTLDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLTVPPLLESVTW IVLKQPINIS SQQLAKFRSL LCTAEGEAAA FLVSNHRPPQ PLKGRKVRAS FHMacaca MSRLSWGYRE HNGPIHWKEF FPIADGDQQS PIEIKTQEVK XP_001095487.1SEQ. ID. NO. 56 mulatta YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVLRGGPLAGSYR LRQFHLHWGS ADDHGSEHIV DGVSYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQKITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLTVPPLLESVIW IVLKQPINVS SQQLAKFRSL LCTAEGEAAA FLLSNHRPPQ PLKGRKVRAS FROryctolagus MSRISWGYGE HNGPIHWNQF FPIADGDQQS PIEIKTKEVK XP_002710714.1SEQ. ID. NO. 57 cuniculus YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVLRGGPLTGNYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEYNSQLQKITDILDSIKE KGKQTRFTNF DPLSLLPSSW DYWTYPGSLTVPPLLESVTW IVLKQPINIS SQQLAKFRSL LCSAEGESAA FLLSNHRPPQ PLKGRKVRAS FHAiluropoda MSRLSWGYGE HNGPIHWNKF FPIADGDQQS PIEIKTKEVK XP_002916937.1SEQ. ID. NO. 58 melanoleuca YDSSLRPLSI KYDANSAKII SNSGHSFSVD FDDTEDKSVLRGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEHNSQLQKITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLTVPPLLESVTW IVLKQPINIS SEQLATFRTL LCTAEGEAAA FLLSNHRPPQ PLKGRKVRAS FHSus scrofa MSRFSWGYGE HNGPVHWNEF FPIADGDQQS PIEIKTKEVK XP_001924497.1SEQ. ID. NO. 59 YDSSLRPLSI KYDPSSAKII SNSGHSFSVD FDDTEDKSVLRGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVKYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGVFLQ IGEHNSQLQKITDILDSIKE KGKQTRFTNF DPLSLLPPSW DYWTYPGSLTVPPLLESVTW IILKQPINIS SQQLATFRTL LCTKEGEEAA FLLSNHRPLQ PLKGRKVRAS FHCallithrix MSRLSWGYGE HNGPIHWNEF FPIADGDRQS PIEIKAKEVK XP_002759085.1SEQ. ID. NO. 60 jacchus YDSSLRPLSI KYDPSSAKII SNSGHSFNVD FDDTEDKSVLHGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELHVVHWNSEKYP SFVEAAHEPD GLAVLGVFLQ IGEPNSQLQKIIDILDSIKE KGKOIRFTNF DPLSLFPPSW DYWTYSGSLTVPPLLESVTW ILLKQPINIS SQQLAKFRSL LCTAEGEAAA FLLSNYRPPQ PLKGRKVRAS FRRattus MARLSWGYDE HNGPIHWNEL FPIADGDQQS PIEIKTKEVK NP_001128465.1SEQ. ID. NO. 61 norvegicus YDSSLRPLSI KYDPASAKII SNSGHSFNVD FDDTEDKSVLRGGPLTGSYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELHVVHWNSDKYP SFVEAAHESD GLAVLGVFLQ IGEHNPQLQKITDILDSIKE KGKQTRFTNF DPLCLLPSSW DYWTYPGSLTVPPLLESVTW IVLKQPISIS SQQLARFRSL LCTAEGESAA FLLSNHRPPQ PLKGRRVRAS FY MusMARLSWGYGE HNGPIHWNEL FPIADGDQQS PIEIKTKEVK NP_078771.1 SEQ. ID. NO. 62musculus YDSSLRPLSI KYDPASAKIISNSGHSFNVD FDDTEDKSVLRGGPLTGNYR LRQFHLHWGS ADDHGSEHVV DGVRYAAELHVVHWNSDKYP SFVEAAHESD GLAVLGVFLQ IGEHNPQLQKITDILDSIKE KGKQTRFTNFDPLCLLPSSW DYWTYPGSLTVPPLLESVTW IVLKQPISIS SQQLARFRSL LCTAEGESAA FLLSNHRPPQ PLKGRRVRAS FYCanis MPPRRHGPNT FLSAGTKGQQ NFWTKNQKSG PIHWNKFFPI XP_544159SEQ. ID. NO. 63 familiaris ADGDQQSPIE IKTKEVKYDS SLRPLSIKYD ANSAKIISNSGHSFSVDFDD TEDKSVLRGG PLTGSYRLRQ FHLHWGSADDHGSEHVVDGV RYAAELHVVH WNSDKYPSFV EAAHEPDGLAVLGVFLQIGE HNSQLQKITD ILDSIKEKGK QTRFTNFDPLSLLPPSWDYW TYPGSLTVPP LLESVTWIVL KQPINISSQQLATFRTLLCT AEGEAAAFLL SNHRPPQPLK GRKVRASFH EguusMSGPVHWNEF FPIADGDQQS PIEIKTKEVK YDSSLRPLTI XP_001489984.2SEQ. ID. NO. 64 caballus KYDPSSAKII SNSGHSFSVG FDDTENKSVL RGGPLTGSYRLRQFHLHWGS ADDHGSEHVV DGVRYAAELH IVHWNSDKYPSFVEAAHEPD GLAVLGVFLQ VGEHNSQLQK ITDTLDSIKEKGKQTLFTNF DPLSLLPPSW DYWTYPGSLT VPPLLESVTWIILKQPINIS SQQLVKFRTL LCTAEGETAA FLLSNHRPPQ PLKGRKVRAS FR Bos taurusMSGFSWGYGE RDGPVHWNEF FPIADGDQQS PIEIKTKEVR XP_002692875.1 SEQ. ID. NO. 65 YDSSLRPLGI KYDASSAKII SNSGHSFNVD FDDTDDKSVLRGGPLTGSYR LRQFHLHWGS TDDHGSEHVV DGVRYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGIFLQ IGEHNPQLQKITDILDSIKE KGKQTRFTNF DPVCLLPPCR DYWTYPGSLTVPPLLESVTW IILKQPINIS SQQLAAFRTL LCSREGETAA FLLSNHRPPQ PLKGRKVRAS FRMonodelphis MSRLSWGYCE HNGPVHWSEL FPIADGDYQS PIEINTKEVK XP_001366749.1SEQ. ID. NO. 66 domestica YDSSLRPLSI KYDPASAKII SNSGHSFSVD FDDSEDKSVLRGGPLIGTYR LRQFHLHWGS TDDQGSEHTV DGMKYAAELHVVHWNSDKYP SFVEAAHEPD GLAVLGIFLQ TGEHNLQMQKITDILDSIKE KGKQIRFTNF DPATLLPQSW DYWTYPGSLTVPPLLESVTW IVLKQPITIS SQQLAKFRSL LYTGEGEAAA FLLSNYRPPQ PLKGRKVRAS FROrnithorhynchus MKKGVGSFYE LAVNRWSVVN RVQIMIVESI TEPLLCGSRAXP_001507177.1 SEQ. ID. NO. 67 anatinusLALTLSPTQA LAVAPALALA VVQALALTVV QALALAVSPALALSVAPALA LAVVQALALA VVQALALAVA QALALAVAQALALAVAQALA LALPQALALT LPQALALTLS PTLALSVAPALALAVAPALA LADSPALALA LARPHPSSGS SPALDCELVLFGDCHTVLLK WMRMGNYSSV SPLEERNSSC PLGPIHWNELFPIADGDRQS PIEIKTKEVK YDSSLRPLSI KYDPTSAKIISNSGHSFSVD FDDTEDKSVL RGGPLSGTYR LRQFHFHWGSADDHGSEHTV DGMEYSAELH VVHWNSDKYS SFVEAAHEPDGLAVLGIFLK RGEHNLQLQK ITDILDAIKE KGKQMRFTNFDPLSLLPLTR DYWTYPGSLT VPPLLESVIW IIFKQPISISSQQLAKFRNL LYTAEGEAAD FMLSNHRPPQ PLKGRKVRAS FRS

TABLE D5Exemplary CA II DNA expression constructs for chloroplast expressionATGTCCCATC ACTGGGGGTA CGGCAAACAC AACGGACCTG AGCACTGGCA TAAGGACTTCSEQ. ID. NO. 94CCCATTGCCA AGGGAGAGCG CCAGTCCCCT GTTGACATCG ACACTCATAC AGCCAAGTAT(human cDNAGACCCTTCCC TGAAGCCCCT GTCTGTTTCC TATGATCAAG CAACTTCCCT GAGGATCCTCsequence)AACAATGGTC ATGCTTTCAA CGTGGAGTTT GATGACTCTC AGGACAAAGC AGTGCTCAAGGGAGGACCCC TGGATGGCAC TTACAGATTG ATTCAGTTTC ACTTTCACTG GGGTTCACTTGATGGACAAG GTTCAGAGCA TACTGTGGAT AAAAAGAAAT ATGCTGCAGA ACTTCACTTGGTTCACTGGA ACACCAAATA TGGGGATTTT GGGAAAGCTG TGCAGCAACC TGATGGACTGGCCGTTCTAG GTATTTTTTT GAAGGTTGGC AGCGCTAAAC CGGGCCTTCA GAAAGTTGTTGATGTGCTGG ATTCCATTAA AACAAAGGGC AAGAGTGCTG ACTTCACTAA CTTCGATCCTCGTGGCCTCC TTCCTGAATC CTTGGATTAC TGGACCTACC CAGGCTCACT GACCACCCCTCCTCTTCTGG AATGTGTGAC CTGGATTGTG CTCAAGGAAC CCATCAGCGT CAGCAGCGAGCAGGTGTTGA AATTCCGTAA ACTTAACTTC AATGGGGAGG GTGAACCCGA AGAACTGATGGTGGACAACT GGCGCCCAGC TCAGCCACTG AAGAACAGGC AAATCAAAGC TTCCTTCAAA TAAgaattcATGTCtCATCAtTGGGGtTAtGGtAAACACAAtGGtCCTGAaCACTGGCATAAaGACTTSEQ. ID. NO. 108tCCaATTGCaAAaGGtGAaCGtCAaTCaCCTGTTGAtATtGACACTCATACAGCtAAaTATGACC(Optimized forCTTCttTaAAaCCatTaTCTGTTTCaTATGATCAAGCAACTTCttTacGtATttTaAACAATGGTchloroplastCATGCTTTtAAtGTaGAaTTTGATGACTCTCAaGAtAAAGCAGTatTaAAaGGtGGtCCatTaGAExpression)TGGtACTTACcGtTTaATTCAaTTTCACTTTCACTGGGGTTCAtTaGATGGtCAAGGTTCAGAaCATACTGTaGATAAAAAaAAATATGCTGCAGAAtTaCACTTaGTTCACTGGAACACaAAATATGGtGATTTTGGtAAAGCTGTaCAaCAACCTGATGGttTaGCtGTTtTAGGTATTTTTTTaAAaGTTGGtAGtGCTAAACCaGGtCTTCAaAAAGTTGTTGATGTatTaGATTCaATTAAAACAAAaGGtAAaAGTGCTGACTTtACTAAtTTCGATCCTCGTGGttTaCTTCCTGAATCtTTaGATTACTGGACaTAtCCAGGtTCAtTaACaACaCCTCCTCTTtTaGAATGTGTaACaTGGATTGTatTaAAaGAACCaATtAGtGTaAGtAGtGAaCAaGTaTTaAAATTCCGTAAACTTAAtTTCAATGGtGAaGGTGAACCaGAAGAAtTaATGGTtGAtAACTGGCGtCCAGCTCAaCCAtTaAAaAAtcGtCAAATtAAAGCTTCaTTCAAATAAgcatgc

TABLE D6 Codons in Human CA II optimized for expression in chloroplastof Chlamydomonas reinhardtii Number of codons No. of amino ExpectedAmino Total that were acids of ratio acid number optimized each codon ofcodons Ser(S) 18 12 TCT TCA 1:1:1 AGT (7:7:5) Phe(F) 12 3 TIT TTC (8:4)2:1 Leu(L) 26 19 TIA CTT (21:5) 5:1 Val(V) 17 10 GTT GTA (8:9) 1:1Pro(P) 17 6 CCT CCA (8:9) 3:4 Thr(T) 12 5 ACT ACA (5:7) 2:3 Ala(A) 13 3GCT GCA (9:4) 2:1 Tyr(Y) 8 2 TAT TAC (6:2) 2:1 His(H) 12 1 CAT CAC (6:6)1:1 Asn(N) 10 4 AAT AAC (7:3) 2.5 1 A(D) 19 3 GAT GAC (14:5) 2.5 1Ile(I) 9 4 ATT (9) 1 Met(M) 2 0 ATG (2) 1 Gln(Q) 11 7 CAA (11) 1 Glu(E)13 6 GAA (13) 1 Lys(K) 24 11 AAA (24) 1 Cys(C) 1 0 TGT (1) 1 Tf£_(W) 7 0TGG (7) 1 Gly(G) 22 17 GGT (22) 1 Arg(R) 7 5 CGT (7) 1

TABLE D7 Exemplary algal bicarbonate transporter types TransportSubstrate Photosynthetic Type Mechanism affinity Flux rate affinity ko.6BicA Na+ Low- High 90-170 μM dependent medium HC0³⁻ SbtA Na+ High Low <5μM HC0₃ dependent HC0³⁻ uptake BicA Na+ Low- High 90-170 μM dependentmedium HC0³⁻ SbtA Na+ High Low <5 μM HC0₃ dependent HC0³⁻ uptake

TABLE D8 Exemplary plasma membrane localized Bicarbonate transportersAccession Organism Sequence Number SEQ. ID. NO ChlamydomonasMLPGLGVILL VLPMQYYFGY KIVQIKLQNA KHVALRSAIM EDP07736.1 SEQ. ID. NO. 77reinhardtii QEVLPAIKLV KYYAWEQFFE NQISKVRREE IRLNFWNCVMKVINVACVFC VPPMTAFVIF TTYEFQRARL VSSVAFTTLSLFNILRFPLV VLPKALRAVS EANASLQRLE AYLLEEVPSGTAAVKTPKNA PPGAVIENGV FHHPSNPNWH LHVPKFEVKPGQVVAVVGRI AAGKSSLVQA ILGNMVKEHG SFNVGGRISYVPQNPWLQNL SLRDNVLFGE QFDENKYTDV IESCALTLDLQILSNGDQSK AGIRGVNFSG GQRQRVNLAR CAYADADLVLLDNALSAVDH HTAHHIFDKC IKGLFSDKAV VLVTHQIEFMPRCDNVAIMD EGRCLYFGKW NEEAQHLLGK LLPITHLLHAAGSQEAPPAP KKKAEDKAGP QKSQSLQLTL APTSIGKPTEKPKDVQKLTA YQAALIYTWY GNLFLVGVCF FFFLAAQCSRQISDFWVRWW VNDEYKKFPV KGEQDSAATT FYCLIYLLLVGLFYIFMIFR GATFLWWVLK SSETIRRKAL HNVLNAPMGFFLVTPVGDLL LNFTKDQDIM DENLPDAVHF MGIYGLILLATTITVSVTIN FFAAFTGALI IMTLIMLSIY LPAATALKKARAVSGGMLVG LVAEVLEGLG VVQAFNKQEY FIEEAARRTNITNSAVFNAE ALNLWLAFWC DFIGACLVGV VSAFAVGMAKDLGGATVGLA FSNIIQMLVF YTWVVRFISE SISLFNSVEGMAYLADYVPH DGVFYDQRQK DGVAKQIVLP DGNIVPAASKVQVVVDDAAL ARWPATGNIR FEDVWMQYRL DAPWALKGVTFKINDGEKVG AVGRTGSGKS TTLLALYRMF ELGKGRILVDGVDIATLSLK RLRTGLSIIP QEPVMFTGTV RSNLDPFGEFKDDAILWEVL KKVGLEDQAQ HAGGLDGQVD GTGGKAWSLGQMQLVCLARA ALRAVPILCL DEATAAMDPH TEAIVQQTIKKVFDDRTTIT IAHRLDTIIE SLMEYESPSK LLANRDSMFSKLVDKTGPAA AAALRKMAED FWSTRSAQGR NQ Volvox carteriMGTISHPARG NDPTAGFFNK EAFGWMFKHV SEARKNGDID XP_002950646.1SEQ. ID. NO. 69 f. nagariensisLDKMGMPPEN HAHEAYDMFA SNWAAEMKLK DSGAKPSLVRALRKSFGLVY LLGGVFKCFW STFVITGAFY FVRSLLAHVNGIKDGRLYSK TVSGWCLMAG FTLDAWLLGL SLQRMGYICMSVGIRARAAL VQAVTHKAFR LSSVRADQSA AIVNFVSSDIQKIYDGALEF HYLWTAPFEA AAILALLGYL TNDSMLPGLGVILLVLPLQY FFGYKIIQIK LQNAKHVALR SSILQEVLPAIKLVKYYAWE QFFEDEISKI RREEMRLSFW NAMMKVINVACVFCVPPMTA FVIFTTYEFQ KARLVSGVAF TTLSLFNILRFPLVVLPKAL RAVSEAHASL QRLESYLLED VPQGTASGGKSSKSSAPGVH IDNAVYHHPS NPNWHLHVPR FDVRPGQVVAVVGRIGAGKS SLVQAILGNM VKEHGSQQVG GRISYVPQNPWLQNLSIRDN VTFGEGWDEN KYEAVIDACA LTMDLQILPQGDQSKAGIRG VNFSGGQRQR VNLARCAYAD ADLVLLDNALSAVDHHTAHH IFDKCIKGLF SDKAVVLITH QIEFMPRCDAVAIMDEGRCL YFGKWNEESQ HLLGKLLPIT HLLHAAGSQEAPPAAPKKKD DKATPQKSQS LQLTLAPTSI GKPTQKDTKAAPKLTAFKAA LIYTYYGNIL LVFVCFITFL AAQTCRQMSDFWVRWWVNDE YKHFPKRTGV REESATKFYA LIYLLLVGLFYFTMVARGST FLWWVLRSSE NIRKKALNNV LNAPMGFFLVTPVGDLLLNF TKDQDIMDEN LPDAIHFMGI YGLILLATTITVSVTINFFG AFTGFLIIMT LIMLAIYLPA ATALKKARAVSGGQLVGLVA EVLEGLNVVQ AFSKQEYFIE EAARRTDVTNAAVFNAESLN LWLAFWCDLI GASLVGVVSA FAVGLKDQLGAATVGLAFSN IIQMLVFYTW VVRFIAESIS LFNSVEAMAWLADYVPKDGI FYDQKQLDGV AKSITLPDGQ IVPATSKVQVVVDDAALARW PATGNIRFED VWMQYRLDAA WALKGVTFKINDGEKVGAVG RTGSGKSTTL LALYRMFELG KGRILIDGVDIATLSLKRLR TGLSIIPQEP VMFTGTVRSN LDPFGEFKDDSVLWEVLQKV GLEAQAQHAG GLDGRVDGTG GKAWSLGQMQLVCLARAALR AVPILCLDEA TAAMDPHTEQ VVQETIKKVFDDRTTITIAH RLDTIIESDK VLVMEAGELK EFAPPAQLLANRETMFSKLV DKTGPAAAAA LRKMADEHFS KSQARAAAQR H ChlorellaMVPLLAQRGR IRSQAPRTWH PDPQPLHAER SRQCPGRGVR EFN52914.1 SEQ. ID. NO. 70variabilis AAAKRGGGSG GATHKSKKSK ELDEVAAFEQ LMCDWDDAFAADCYDNERAA RMARLAEEGY QHHGRGFVFV RSRLDKRSRKARNDSGASKG FGAAAKALSV EQGTPLENNP QLHLLSWTACYIASSQLDSL GGLFSTQEGV LLPDSGSLLT DGGSGASGSNAADAVGELQR VLRGQDLSQL RGYVGAPPQA RPASGSDDDGSSTTGSNNGA AGEGSEVEEG TAMGGIRRYE PESGELVVLLSCKIGGKPAV GAELLAVAQA EDGKHAPGAS PDTRLCKEPSQSAFDLWSFG WMNKIVPAAR RGEVEVADLP LPEAQQAEPCYEELNTNWEA AVQEAKKAGK EPKLMKVLWK TYGKDIVLAGIFKLMWSVFV ILGAYYFTRS ILMCIRTLEG KDDSIYDTEWKGWVLTGFFF LDAWLLGMML QRMAFNCLKV GIKARAALTTMIARKCYNMA HLTKDTAAEA VGFVASDINK VFEGIQEVHYLWGAPVEAGA ILALLGTLVG VYCIGGVIIV CMVVPLQYYFGYKIIKNKIK NAPNVTERWS IIQEILPAMK LVKYYAWERFFEKHVADMRT RERHYMFWNA VVKTVNVTMV FGVPPMVTFAVLVPYELWHV DSSTSEPYIK PQTAFTMLSL FNVLRFPLVVLPKAMRCVSE ALRSVGNLEK FLAEPVAPRQ DLEGKPGAQLSKAVLRHEMD TSGFTLRVPE FSVKAGELVA VVGRVGAGKSSILQAMLGNM QTASGLAKCQ HSASSCLPFL VEGTAHSGGRIAYVPQTAWC QNLSLRDNIT FGQPWDEAKY KQVIHACALELDLAILAAGD QSKAGLRGIN LSGGQRQRLN LARCAYFDGDLVLLDNALSA VDHHTAHHIF EHCVRGMFRD KATVLVTHQVEFLPQCDKVA IMDDGTCVYF GPWNAAAQQL LSKYLPASHLLAAGGNAEQP RDTKKKVVKK EETKKTEDAG KAKRVHSASLTLKSALWEYC WDARWIIFCL SLFFFLTAQA SRQLADYFIRWWTRDHYNKY GVLCIDEGDN PCGPLFYVQY YGILGLLCFIVLMAFRGAFL YTWSLGASYR QHEKSIHRVL YAPLGFFLTTPVGDLLVSFT KDQDVMDDAL PDALYYAGIY GLILLATAITVSVTIPLFSA LAGGLFVVSG IMLAIYLPAA THLKKLRMGTSGDVVTLIAE ALDGLGVIQA YGKQAYFTTI TSQYVNDAHRALFGAESLNL WLAFICDFFG ACMVLSVACF GIGQWSTLGSSSVGLAFSQS IQMLVFYTWS IRLVAECIGL FGSAEKIAWLANHTPQEAGS LDPPSLPGSG ETKAAPKKRG TAGKFLPPLKDEDLAIVPTG GPKLPSGWPR TGVLEFNQVV MKYAPHLPPALRGVSFKVKS GDKVGVVGRT GSGKSTLLLA LYRMFNLESGAITLDGIDIS TLTLEQLRRG LSVIPQEPTV FSGTVRTNLDPFGEFGADAI LWEALRDCGL EEQVKACGGL DAKLDGTGGNAWSIGQQQLM CLARAALKKV PVLCLDEATA AMDPHTEAHVLEIIERIFSD RTMLTIAHRL DNVIRSDLVV VMDAGQVCEMGTPDELLANP_QSAFSQLVDK TGAASAAALR KMAADFLDERARGQKLGFKP RPSLEESHIC VAPSPSLILS TLLFPPAFMANVTALLLPKP VLSHAPVSSQ TVNTYIRLNI IQLQCNVLHPATKEATWSSR RITFTAHLSS SGSKPPPPLP PLTELPEGRGLDWSSAGYRD GREAIPSPSA KYSAADYGAA GDGVTDDTQALQVAVAAAHE DDEGGVVYLG AGTFVLTQPL SIAGSNVVIRGAGEDATTIF VPLPLSDVFP GTWSMDASGK VTSPWITRGGFLAFSGRRTK SSDSSTLLAT VAGSVEQGAS VIPVDSTAEFRLGQWVRIII NDASTDASAG GGTLERGSSE VQESETMIAEGATGGGAGVR AQWTGVLHAF EPTVQCSGVE QLTIRFNHSMMAAHLAERGY NAIELEDVVD CWIRQVTILN ADNAIRLRGTDHSTLSGQAC SGGGVVAVVP VWCRRGLPSP ADVTVGVTELRWEPDTREVN GHHAITVSKG HANLVTRFRI TAPFYHDISLEGGALLNVIS SGGGANLNLD LHRSGPWGNL FSQLGMGLAARPFDAGGRDG RGAHAGRQNT FWNLQPGDVA AAAPALQPSAAAGDARRLLV DGDSLLHAGT GQARLLRQLE ADDSAEPLLLPSCEFGPLLN FVGGFAGELC KSSGWLVAGL PDDRPDLHAS QVTARLQHGA ADNKTHASynechococcus MDFLSNFLMD FVKQLQSPTL SFLIGGMVIA ACGSQLQIPE ABB57505.1SEQ. ID. No. 71 elongatus SICKIIVFML LTKIGLTGGM AIRNSNLTEM VLPALFSVAIPCC 7942.J GILIVFIARY TLARMPKVKT VDAIATGGLF GAVSGSTMAAALTLLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLNKKKRKEAAFA SAQGAYSKQP VAAGDYSSSS DYPSSRREYAQQESGDHRVK IWPIVEESLQ GPALSAMLLG VALGLFARPESVYEGFYDPL FRGLLSILML VMGMEAWSRI SELRKVAQWYVVYSIVAPLA HGFIAFGLGM IAHYATGFSM GGVVVLAVIAASSSDISGPP TLRAGIPSAN PSAYIGASTA IGTPVAIGIA IPLFLGLAQT IGG SynechocystisMDFLSNFLTD FVGQLQSPTL AFLIGGMVIA ALGTQLVIPE NP_441340 SEQ. ID. No.72 sp.AISTIIVFML LTKIGLTGGM AIRNSNLTEM LLPVAFSVIL PCC 6803GILIVFIARF TLAKLPNVRT VDALATGGLF GAVSGSTMAAALTTLEESKI SYEAWAGALY PFMDIPALVT AIVVANIYLNKRKRKSAAAS IEESFSKQPV AAGDYGDQTD YPRTRQEYLSQQEPEDNRVK IWPIIEESLQ GPALSAMLLG LALGIFTKPESVYEGFYDPL FRGLLSILML IMGMEAWSRI GELRKVAQWYVVYSLIAPIV HGFIAFGLGM IAHYATGFSL GGVVVLAVIAASSSDISGPP TLRAGIPSAN PSAYIGSSTA IGTPIAIGVC IPLFIGLAQT LGAG Nostoc sp.MDFFSLFLMD FVKQLQSPTL GFLIGGMVIA ALGSELIIPE NP_486174 SEQ. ID. No. 73PCC 712 AICQIIVFML LTKIGLTGGI AIRNSNLTEM VLPAASAVAVGVLVVFIARY TLAKLPKVNT VDAIATGGLF GAVSGSTMAAALTLLEEQKI QYEAWAAALY PFMDIPALVT AIVVANIYLNKKKRSAAGEY LSKQSVAAGE YPDQQDYPSS RQEYLRKQQSADNRVKIWPI VKESLQGPAL SAMLLGIALG LFTQPESVYKSFYDPLFRGL LSILMLVMGM EAWSRIGELR KVAQWYVVYSVVAPLVHGFI AFGLGMIAHY ATGFSLGGVV ILAVIAASSSDISGPPTLRA GIPSANPSAY IGASTAIGTP IAIGLAIPLF LGLAQAIGGR Cyanothece sp.MDFWSYFLMD FVKQLQSPTL GFLIGGMVIA ALGSQLVIPE YP_002485721 SEQ. ID. No. 74PCC 7425 AICQIIVFML LTKIGLTGGM AIRNSNLTEM VLPAAFSVISGILIVFIARY TLAKLPKVRT VDAIATGGLF GAVSGSTMAAALTLLEEEKI PYEAWAGALY PFMDIPALVT AIVIANIYLNKKKRRAESEA LSKQEYLGKQ SIVAGDYPAQ QDYPSTRQEYLSKQQGPENN RVKIWPIVQE SLQGPALSAM LLGVALGILTKPESVYESFY DPLFRGLLSI LMLVMGMEAW SRIGELRKVAQWYVVYSVVA PFVHGLIAFG LGMFAHYTMG FSMGGVVVLAVIASSSSDIS GPPTLRAGIP SANPSAYIGA STAIGTPIAI GLCIPFFIGL AQTLGGGMicrocysti MDFFSLFVMD FIQQLQSPTL AFLIGGMIIA ALGSELVIPE YP_001661223SEQ. ID. No. 75 aeruginosa SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMIFAVIVNIES-843 GIIVVFVARY TLANLPKVKV VDAIATGGLF GAVSGSTMAAGLTVLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLNKKKQKEAAYD QESFSKQPVA AGNYSDQQDY PSSRQEYLSQQQPADNRVKI WPIIEESLRG PALSAMLLGL ALGIFTQPESVYKSFYDPLF RGLLSVLMLV MGMEAWSRVG ELRKVAQWYVVYSVIAPFVH GLIAFGLGMI AHYATGFSWG GVVMLAVIASSSSDISGPPT LRAGIPSANP SAYIGASTAI GTPVAIGLCI PFFVGLAQAL SGG AnabaenaMDFVSLFVKD FIAQLQSPTL AFLIGGMIIA ALGSELVIPE YP_323532 SEQ. ID. No. 86variabills SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMIFAVIT ATCC 29413GITIVFISRY TLAKLPKVKV VDAIATGGLF GAVSGSTMAAGLTVLEEQKM AYEAWAGALY PFMDIPALVT AIVIANIYLNKKKRKEAVYS TEQPVAAGDY PDQKDYPSSR QEYLSQQKGDEDNRVKIWPI IEESLRGPAL SAMLLGLALG LFTQPESVYKSFYDPAFRGL LSILMLVMGM EAWSRIGELR KVAQWYVVYSVVAPFVHGLI AFGLGMIAHY TMNFSMGGVV ILAVIASSSSDISGPPTLRA GIPSANPSAY IGASTAVGTP VAIGLCIPFF LGLAQAIGG Cyanothece sp.MDFLSLFVKD FIIQLQSPTL AFLIGGMVIA ALGSELVIPE YP_002371470.1SEQ. ID. No. 87 PCC 880 SICTIIVFML LTKIGLTGGI AIRNSNLTEM VLPMICAVIV  GIVVVFIARY TLAKLPKVNV VDAIATGGLF GAVSGSTMAAGLTVLEEQKI PYEAWAGALY PFMDIPALVT AIVVANIYLNKKKRKATVMQ ESLSKQPVAA GDYPSSRQEY VSQQQPEDNRVKIWPIIEES LRGPALSAML LGLALGILTQ PESVYKGFYDPPFRGLLSIL MLVMGMEAWS RIGELRKVAQ WYVVYSVAAPFIHGLLAFGL GMIAHYTMGF SMGGVVILAV IASSSSDISGPPTLRAGIPS ANPSAYIGAS TAIGTPVAIG LCIPFFVGLA QAIGGF ArthrospiaMDFLSGFLTR FLAQLQSPTL GFLIGGMVIA AVNSQLQIPD ZP.06383808.1SEQ. ID. No. 88  platensis AIYKFVVFML LMKVGLSGGI AIRGSNLTEM LLPAVFALVTstr. GIVIVFIGRY TLAKLPNVKT VDAIATAGLF GAVSGSTMAA ParacaALTLLEEQGM EYEAWAAALY PFMDIPALVS AIVLASIYVSKQKHSDMADE SLSKHESLSK QPVAAGDYPS KPEYPTTRQEYLSQQRGSAN QGVEIWPIIK ESLQGSALSA LLLGLALGLLTRPESVFQSF YEPLFRGLLS ILMLVMGMEA TARLGELRKVAQWYAVYAFI APLLHGLIAF GLGMIAHVVT GFSLGGVVILAVIASSSSDI SGPPTLRAGI PSANPSAYIG SSTAVGTPVA IALGIPLYIG LAQALMGG

TABLE D9Exemplary chloroplast envelope localized Bicarbonate transportersAccession Organism Sequence Number SEQ. ID. NOMQTTMTRPCL AQPVLRSRVL RSPMRVVAAS APTAVTTVVT BAD16681.1 SEQ. ID. NO.89Chlamydomonas SNGNGNGHFQ AATTPVPPTP APVAVSAPVR AVSVLTPPQV reinhardtiiYENAINVGAY KAGLTPLATF VQGIQAGAYI AFGAFLAISVGGNIPGVAAA NPGLAKLLFA LVFPVGLSMV TNCGAELFTGNTMMLTCALI EKKATWGQLL KNWSVSYFGN FVGSIAMVAAVVATGCLTTN TLPVQMATLK ANLGFTEVLS RSILCNWLVCCAVWSASAAT SLPGRILALW PCITAFVAIG LEHSVANMFVIPLGMMLGAE VTWSQFFFNN LIPVTLGNTI AGVLMMAIAY SISFGSLGKS AKPATAVolvox carteri MQTTMSVTRP CVGLRPLPVR NVRSLIRAQA APQQVSTAVSXP_002951507.1 SEQ. ID. NO. 79 f. nagariensisTNGNGNGVAA ASLSVPAPVA APAQAVSTPV RAVSVLTPPQVYENAANVGA YKASLGVLAT FVQGIQAGAY IAFGAFLACSVGGNIPGITA SNPGLAKLLF ALVFPVGLSM VTNCGAELYTGNTMMLTCAI FEKKATWAQL VKNWVVSYAG NFVGSIAMVAAVVATGLMAS NQLPVNMATA KSSLGFTEVL SRSILCNWLVCCAVWSASAA TSLPGRILGL WPPITAFVAI GLEHSVANMFVIPLGMMLGA DVTWSQFFFN NLVPVTLGNT IAGVVMMAVA YSVSYGSLGK TPKPATA

TABLE D10 Transit Peptides Organism SEQ ID NO Name Arabidopsis 8 Rbcs-1atransit thaliana peptide Arabidopsis 14 PGR5 transit thaliana peptideArabidopsis 15 psaD transit thaliana peptide Arabidopsis 22 DNAJ transitthaliana peptide Cyanophora 102 psaD trasit paradoxa peptide Arabidopsis104 CAB transit thaliana peptide Arabidopsis 105 PGR5 transit thalianapeptide

TABLE D11 Cyclic Electron Transfer modulator proteins SEQ ID AccessionOrganism NO Name No. Function Arabidopsis 93 Ferredoxin1 AEE28669.1cyclic electron thaliana (FD1) transfer modulator protein Arabidopsis 95Ferredoxin2 AAG40057.1 cyclic electron thaliana (FD2) transfer modulatorprotein Arabidopsis 96 ferredoxin- AT5G66190 cyclic electron thalianaNADP(+) partial transfer oxidoreductase modulator (FNR1) proteinArabidopsis 97 ferredoxin- BAH19611.1 cyclic electron thaliana NADP(+)transfer oxidoreductase modulator (FNR2) protein

An exemplary optimized DNA sequence for the plasma membrane localizedbicarbonate transporter is shown in SEQ ID NO. 91

(SEQ ID NO: 91)atgctgcccg gcctgggcgt catcctgctg gtgctgccca tgcagtacta cttcggctac 60aagatcgtgc agatcaagct gcagaacgcc aagcacgtcg ccctgcgctc cgccatcatg 120caggaggtgc tgcccgccat caagctggtc aagtactacg cctgggagca gttctttgag 180aaccagatca gcaaggtccg ccgcgaggag atccgcctca acttctggaa ctgcgtgatg 240aaggtcatca acgtggcctg cgtgttctgc gtgccgccca tgaccgcctt cgtcatcttc 300accacctacg agttccagcg cgcccgcctg gtgtccagcg tcgccttcac caccctgtcg 360ctgttcaaca ttctgcgctt ccccctggtc gtgctgccca aggccctgcg tgccgtgtcc 420gaggccaacg cgtctctcca gcgcctggag gcctacctgc tggaggaggt gccctcgggc 480actgccgccg tcaagacccc caagaacgct ccccccggcg ccgtcatcga gaacggtgtg 540ttccaccacc cctccaaccc caactggcac ctgcacgtgc ccaagttcga ggtcaagccc 600ggccaggtcg ttgctgtggt gggccgcatc gccgccggca agtcgtccct ggtgcaggcc 660atcctcggca acatggtcaa ggagcacggc agcttcaacg tgggcggccg catctcctac 720gtgccgcaga acccctggct gcagaacctg tccctgcgtg acaacgtgct gtttggcgag 780cagttcgatg agaacaagta caccgacgtc atcgagtcct gcgccctgac cctggacctg 840cagatcctgt ccaacggtga ccagtccaag gccggcatcc gcggtgtcaa cttctccggt 900ggccagcgcc agcgcgtgaa cctggcccgc tgcgcctacg ccgacgccga cctggtgctg 960ctcgacaacg ccctgtccgc cgtggaccac cacaccgccc accacatctt cgacaagtgc 1020atcaagggcc tgttctccga caaggccgtg gtgctggtca cccaccagat cgagttcatg 1080ccccgctgcg acaacgtggc catcatggac gagggccgct gcctgtactt cggcaagtgg 1140aacgaggagg cccagcacct gctcggcaag ctgctgccca tcacccacct gctgcacgcc 1200gccggctccc aggaggctcc ccccgccccc aagaagaagg ccgaggacaa ggccggcccc 1260cagaagtcgc agtcgctgca gctgaccctg gcccccacct ccatcggcaa gcccaccgag 1320aagcccaagg acgtccagaa gctgactgcc taccaggccg ccctcatcta cacctggtac 1380ggcaacctgt tcctggttgg cgtgtgcttc ttcttcttcc tggcggctca gtgctctcgc 1440cagatctccg atttctgggt gcgctggtgg gtgaacgacg agtacaagaa gttccccgtg 1500aagggcgagc aggactcggc cgccaccacc ttctactgcc tcatctacct gctgctggtg 1560ggcctgttct acatcttcat gatcttccgc ggcgccactt tcctgtggtg ggtgctcaag 1620tcctcggaga ccatccgcag gaaggccctg cacaacgtcc tcaacgcgcc catgggcttc 1680ttcctggtca cgccggtcgg cgacctgctg ctcaacttca ccaaggacca ggacattatg 1740gatgagaacc tgcccgatgc cgttcacttc atgggcatct acggcctgat tctgctggcg 1800accaccatca ccgtgtccgt caccatcaac ttcttcgccg ccttcaccgg cgcgctgatc 1860atcatgaccc tcatcatgct ctccatctac ctgcccgccg ccactgccct gaagaaggcg 1920cgcgccgtgt ctggcggcat gctggtcggc ctggttgccg aggttctgga gggccttggc 1980gtggttcagg ccttcaacaa gcaggagtac ttcattgagg aggccgcccg ccgcaccaac 2040atcaccaact ccgccgtctt caacgccgag gcgctgaacc tgtggctggc tttctggtgc 2100gacttcatcg gcgcctgcct ggtgggcgtg gtgtccgcct tcgccgtggg catggccaag 2160gacctgggcg gcgcgaccgt cggcctggcc ttctccaaca tcattcagat gcttgtgttc 2220tacacctggg tggtccgctt catctccgag tccatctccc tcttcaactc cgtcgagggc 2280atggcctacc tcgccgacta cgtgccccac gatggtgtct tctatgacca gcgccagaag 2340gacggcgtcg ccaagcaaat cgtcctgccc gacggcaaca tcgtgcccgc cgcctccaag 2400gtccaggtcg tggttgacga cgccgccctc gcccgctggc ctgccaccgg caacatccgc 2460ttcgaggacg tgtggatgca gtaccgcctg gacgctcctt gggctctgaa gggcgtcacc 2520ttcaagatca acgacggcga gaaggtcggc gccgtgggcc gcaccggctc cggcaagtcc 2580accacgctgc tggcgctgta ccgcatgttc gagctgggca agggccgcat cctggtcgac 2640ggcgtggaca tcgccaccct gtcgctcaag cgcctgcgca ccggcctgtc catcattccc 2700caggagcccg tcatgttcac cggcaccgtg cgctccaacc tggacccctt cggcgagttc 2760aaggacgatg ccattctgtg ggaggtgctg aagaaggtcg gcctcgagga ccaggcgcag 2820cacgccggcg gcctggacgg ccaggtcgat ggcaccggcg gcaaggcctg gtctctgggc 2880cagatgcagc tggtgtgcct ggctcgcgcc gccctgcgcg ccgtgcccat cctgtgcctg 2940gacgaggcta ccgccgccat ggacccgcac actgaggcca tcgtgcagca gaccatcaag 3000aaggtgttcg acgaccgcac caccatcacc attgcccacc gcctggacac catcatcgag 3060tccgacaaga tcatcgtgat ggagcagggc tcgctgatgg agtacgagtc gccctcgaag 3120ctgctcgcca accgcgactc catgttctcc aagctggtcg acaagaccgg ccccgccgcc 3180gccgctgcgc tgcgcaagat ggccgaggac ttctggtcca ctcgctccgc gcagggccgc 3240aaccagtaa

An exemplary optimized DNA sequence for Chloroplast envelope localizedBicarbonate transporter is shown in SEQ ID NO: 92

(SEQ ID NO: 92)atgcagacca ctatgactcg cccttgcctt gcccagcccg tgctgcgatc tcgtgtgctc 60cggtcgccta tgcgggtggt tgcagcgagc gctcctaccg cggtgacgac agtcgtgacc 120tcgaatggaa atggcaacgg tcatttccaa gctgctacta cgcccgtgcc ccctactccc 180gctcccgtcg ctgtttccgc gcctgtgcgc gctgtgtcgg tgctgactcc tcctcaagtg 240tatgagaacg ccattaatgt tggcgcctac aaggccgggc taacgcctct ggcaacgttt 300gtccagggca tccaagccgg tgcctacatt gcgttcggcg ccttcctcgc catctccgtg 360ggaggcaaca tccccggcgt cgccgccgcc aaccccggcc tggccaagct gctatttgct 420ctggtgttcc ccgtgggtct gtccatggtg accaactgcg gcgccgagct gttcacgggc 480aacaccatga tgctcacatg cgcgctcatc gagaagaagg ccacttgggg gcagcttctg 540aagaactgga gcgtgtccta cttcggcaac ttcgtgggct ccatcgccat ggtcgccgcc 600gtggtggcca ccggctgcct gaccaccaac accctgcctg tgcagatggc caccctcaag 660gccaacctgg gcttcaccga ggtgctgtcg cgctccatcc tgtgcaactg gctggtgtgc 720tgcgccgtgt ggtccgcctc cgccgccacc tcgctgcccg gccgcatcct ggcgctgtgg 780ccctgcatca ccgccttcgt ggccatcggc ctggagcact ccgtcgccaa catgttcgtg 840attcctctgg gcatgatgct gggcgctgag gtcacgtgga gccagttctt tttcaacaac 900ctgatccccg tcaccctggg caacaccatt gctggcgttc tcatgatggc catcgcctac 960tccatctcgt tcggctccct cggcaagtcc gccaagcccg ccaccgcg 1008

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the disclosure specifically described herein. For examplea transgenic plant or alga of an embodiment disclosed herein furthercomprising within its genome, and expressing or overexpressing, acombination of heterologous nucleotide sequences encoding additionally aRubisco (for example SEQ ID NO:107). Further still a transit peptideamino acid sequence at the amine terminal portion of a protein sequenceidentified herein may be cleaved leaving the protein sequence alone. Thepercent homology applies to the protein sequence without the transitpeptide sequence also. Such equivalents are intended to be encompassedwithin the scope of the following claims.

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What is claimed is:
 1. A construct comprising: i) a first heterologousnucleic acid sequence comprising a first heterologous polynucleotidesequence encoding a cyclic electron modulator gene wherein the cyclicelectron modulator is operatively linked to at least one regulatoryelement wherein the first heterologous nucleic acid sequence encodes aprotein having a sequence selected from PRG5 (SEQ ID NO: 2) or PGRL1(SEQ ID NO:3) or a sequence with at least 80% sequence homology thereto;and ii) a second heterologous nucleic acid sequence comprising a secondheterologous polynucleotide sequence encoding an ATP dependentbicarbonate anion transporter localized to the plasma membrane whereinthe ATP dependent bicarbonate anion transporter localized to the plasmamembrane is operatively coupled to the at least one regulatory elementwherein the second heterologous nucleic acid sequence is HLA3 (SEQ IDNO:77) or a sequence with at least 80% sequence homology thereto.
 2. Theconstruct of claim 1 wherein the HLA3 is codon optimized for plantexpression.
 3. The construct of claim 1 wherein the at least oneregulatory element includes a promoter.
 4. The construct of claim 1wherein the at least one regulatory element is a tissue specificpromoter.
 5. The construct of claim 1 wherein the at least oneregulatory element includes a promoter that is a greentissue/leaf-specific promoter.
 6. The construct of claim 1 wherein thepromoter is selected from among CAB and rbcS.
 7. The construct of claim1 wherein the first nucleic acid sequence and the second nucleic acidsequence encode: i) the PGR5 protein, and the HLA3 protein; or ii) thePGRL1 protein, and the HLA3 protein.
 8. The construct of claim 1 furthercomprising iii) a third heterologous nucleic acid sequence comprising athird heterologous polynucleotide sequence encoding a bicarbonate aniontransporter protein localized to the chloroplast envelope wherein thebicarbonate anion transporter protein localized to the chloroplastenvelope is operatively coupled to the regulatory element wherein thethird heterologous nucleic acid sequence is LCIA (SEQ ID NO:18) or asequence with at least 80% sequence homology thereto.
 9. The constructof claim 8 wherein the at least one regulatory element is a greentissue/leaf-specific promoter.
 10. The construct of claim 8 wherein theat least one regulatory element includes a promoter selected from amongCAB and rbcS.
 11. The construct of claim 1 further comprising iii) athird heterologous nucleic acid sequence comprising a third heterologouspolynucleotide sequence encoding a carbonic anhydrase wherein thecarbonic anhydrase is operatively coupled to the at least one regulatoryelement wherein the third heterologous nucleic acid sequence encodes aprotein selected from a human carbonic anhydrase-2 (HCA2) (SEQ ID NO:17)or a bacterial Neisseria gonorrhoeae carbonic anhydrase (BCA) (SEQ IDNO: 4) or a sequence with at least 80% sequence homology thereto. 12.The construct of claim 11 wherein the at least one regulatory elementincludes a green tissue/leaf-specific promoter.
 13. The construct ofclaim 11 wherein the at least one regulatory element includes a promoterselected from among CAB and rbcS.
 14. The construct of claim 11 whereinthe third heterologous nucleic acid sequence encodes the BCA protein(SEQ ID NO: 4) protein or a sequence with at least 80% sequence homologythereto.
 15. The construct of claim 8 wherein the heterologousnucleotide sequences encode the PGR5 protein, the HLA3 protein, and theLCIA protein or sequences with at least 80% homology thereto.
 16. Theconstruct of claim 11 wherein the heterologous nucleotide sequencesencode the PGR5 protein, the HLA3 protein, and the BCA protein orsequences with at least 80% homology thereto.
 17. The construct of claim11, wherein a) the PGR5 protein has an amino acid sequence at least 80%identical to SEQ ID NO:1; b) the HLA3 protein has an amino acid sequenceat least 80% identical to SEQ ID NO:77; and c) the BCA protein has anamino acid sequence at least 80% identical to SEQ ID NO:21.
 18. Theconstruct of claim 1 wherein the at least one regulatory element of thefirst heterologous nucleic acid sequence and the at least one regulatoryelement of the second heterologous nucleic acid sequence includes apromoter which can be the same or different for the first heterologousnucleic acid and the second heterologous nucleic acid.
 19. A seedcomprising the construct of claim
 1. 20. A vector comprising theconstruct of claim 1.